Antibiotic compositions

ABSTRACT

Disclosed herein are antibiotic compositions, for example compositions that comprise a metal-containing agent and an organoselenium agent, and uses thereof.

CROSS-REFERENCE

This application is a continuation of U.S. application Ser. No.15/832,045 filed Dec. 5, 2017, now U.S. Pat. No. 10,653,717 issued May19, 2020, which claims the benefit of U.S. Provisional Application No.62/430,101 filed Dec. 5, 2016, which is incorporated herein by referencein its entirety.

BACKGROUND

Antibiotic resistance has become a great challenge all over the world.Gram-negative bacteria present a major threat to human life andmedicine, with almost no antibiotics left for treatment making it urgentto find new principles and mechanisms. A concerted focus since the 1990son tackling rising multidrug-resistant (MDR) Gram-positive bacteriawithin US and European healthcare systems appears to have beeninstrumental in stimulating the relatively large numbers of productstargeting Gram-positive bacteria in recent years. The emergence of MDRGram-negative bacteria presents a great threat to human life and is achallenge for modern medicine.

BRIEF SUMMARY

In some cases, the present disclosure provides an antibioticcomposition, wherein the antibiotic composition comprises: asilver-containing agent; and an organoselenium agent. In some instances,the silver-containing agent can comprise a silver ion. In someinstances, the silver-containing agent can comprise silver nitrate. Insome instances, the silver-containing agent can comprise silverdihydrogen citrate. The sliver-containing agent can be provided assilver ions, silver nitrate and/or silver dihydrogen citrate. In someinstances, the organoselenium agent can comprise a selenazol compound.In some instances, the organoselenium agent can comprise abenzoisoselenazol-3(2H)-one compound. In some instances, theorganoselenium agent can comprise an ebselen. The organoseleniumcompound can comprise a selenazol compound, abenzoisoselenazol-3(2H)-one compound, and/or an ebselen. In someinstances, the antibiotic composition can be in a dosage form of liquid.In some instances, the antibiotic composition can be in a dosage form ofa solution or a suspension. In some instances, a concentration of thesilver-containing agent in the antibiotic composition can be about 0.5to 50 μM, about 1 to 25 μM, or about 1 to 10 μM. In some instances, aconcentration of the silver-containing agent in the antibioticcomposition can be about 5 μM. In some instances, a concentration of theorganoselenium agent in the antibiotic composition can be about 4 to 25μM, about 30 to 200 μM, about 30 to 150 μM, or about 30 to 100 μM. Insome instances, a concentration of the organoselenium agent in theantibiotic composition can be about 40 μM or about 80 μM. In someinstances, the silver-containing agent to the organoselenium agent orthe reverse can be a molar ratio of about 1:2 to about 1:20, for exampleabout 1:4, 1:8, or 1:16. In some instances, the antibiotic compositionexhibits an IC₅₀ value of about 10-100 nM to one or more Gram-negativebacteria. In some instances, the antibiotic composition exhibits an IC₅₀value of about 10-100 nM to one or more Gram-positive bacteria. In someinstances, the antibiotic composition exhibits an IC₅₀ value of about 50nM or lower to one or more Gram-negative bacteria. In some instances,the one or more Gram-negative bacteria can comprise K. pneumonia, A.baumannii, P. aeruginosa, E. cloacae, E. coli, or any combinationthereof. In some instances, the antibiotic composition can compriseAgNO₃ and ebselen. In some instances, the antibiotic composition cancomprise 5 μM of AgNO₃ and 4 μM of ebselen in a liquid dosage form. Insome instances, the antibiotic composition can comprise 5 μM of AgNO₃and 20 μM of ebselen in a liquid dosage form. In some instances, theantibiotic composition can comprise 5 μM of AgNO₃ and 40 μM of ebselenin a liquid dosage form. In some instances, the antibiotic compositioncan comprise 5 μM of AgNO₃ and 80 μM of ebselen in a liquid dosage form.

In some cases, the present disclosure provides a pharmaceuticalformulation that can comprise the antibiotic composition disclosedherein. In some instances, the pharmaceutical formulation can furthercomprise an excipient disclosed herein.

In some cases, the present disclosure provides a method of inhibiting orkilling one or more bacteria, comprising contacting the antibioticcomposition disclosed herein with the one or more bacteria. In somecases, the present disclosure provides a method of treating a bacterialinfection, comprising contacting the antibiotic composition disclosedherein with the bacterial infection. In some instances, the one or morebacteria comprise one or more Gram-negative bacteria. In some instances,the one or more bacteria comprise one or more Gram-positive bacteria. Insome instances, the one or more bacteria comprise one or moremultidrug-resistant bacteria. In some instances, the one or morebacteria comprise one or more multidrug-resistance Gram-negativebacteria. In some instances, the one or more bacteria can comprise K.pneumonia, A. baumannii, P. aeruginosa, E. cloacae, E. coli, or anycombination thereof. In some instances, the bacterial infection or oneor more bacteria can be on a surface. In some instances, the bacterialinfection or one or more bacteria can be in a mammal. In some instances,the bacterial infection or one or more bacteria can be in a human. Insome instances, the contacting can be by injection, for exampleintravenous or subcutaneous injection. In some instances, the contactingcan be by topical application. In some instances, the contacting can beby oral administration. In some instances, the contacting lasts for atleast about: 1 minute, 2 minute, 3 minutes, 4 minutes, 5 minutes, 10minutes, 20 minutes, 30 minute, 40 minutes, 50 minutes, 1 hour, 2 hours,3 hours, 4 hours, 5 hours, 6 hours, 7 hour, 8 hours, 9 hours, 10 hours,11 hours, 12 hours, 18 hours, one day, two days, three days, four days,five days, six days, one week, or one month. In some instances, thecontacting occurs 1, 2, 3, 4, 5, 6, 7, or 8 times hourly or daily. Insome instances, the contacting occurs about every 3, 4, 5, 6, 7, 8, 9,10, 11, or 12 minutes or hours daily. In some instances, the antibioticcomposition can be in a single unit dose. In some instances, the amountof the organoselenium agent contacted with the bacterial infection orone or more bacteria can be about 10 to 100 mg, about 10 to 50 mg, orabout 20 to 30 mg, for example about 25 mg, per dosage. In someinstances, an amount of the silver contacted with the bacterialinfection or one or more bacteria can be about 1 to 20 mg, about 1 to 10mg, or about 5 to 7 mg, for example about 6 mg, per dosage.

In some cases, the present disclosure provides a method of making anantibiotic composition, comprising mixing a silver-containing agent andan organoselenium agent. In some instances, the mixing can be conductedin a liquid. In some instances, the mixing can comprise adding thesilver-containing agent to a liquid that can comprise the organoseleniumagent. In some instances, the mixing can comprise adding theorganoselenium agent to a liquid that can comprise the silver-containingagent.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B illustrate effects of silver with ebselen in combinationon the growth of E. coli and HeLa Cells. FIG. 1A is a line chart showingsynergistic effect of ebselen with silver nitrate (AgNO3) in combinationon the growth of E. coli. E. coli DHB4 overnight cultures were diluted1:1000 into 100 μl of LB medium in 96 micro-well plates, and treated by100 μl serial dilutions of ebselen and AgNO3 in combination for 16 h,and cell viability was determined by measuring OD_(600 nm). Ag⁺ aloneinhibited E. coli growth with a minimal inhibition concentration (MIC)of 42 μM after 16 h treatment, while 2 μM ebselen dramatically decreasedthe MIC of Ag⁺ to 4.2 μM (p=0.000028<0.001). FIG. 1B is a line chartshowing effects of ebselen with AgNO3 in combination on the growth ofHeLa cells. HeLa cells were treated with serial concentrations ofebselen and AgNO3 for 24 h, and cell toxicity was detected by MTT assay.5.0 μM Ag⁺ and 2.5 μM ebselen in combination showed no synergistictoxicity on human HeLa cells (p=0.98>0.05). In FIGS. 1A and 1B, data arepresented as means±s. d. of three independent experiments. *: p<0.05,**: p<0.01, ***: p<0.001 (t-test). FIG. 1C is a bar chart showing thatebselen alone has no effect on the growth of E. coli. E. coli DHB4overnight cultures were diluted 1:1000 into 100 μl of LB medium in 96micro-well plates and treated with different concentrations of ebselenfor 16 h. The cell viability was determined by measuring the absorbanceat 600 nm. Data are presented as means±s. d. of three independentexperiments. *: p<0.05, **: p<0.01, ***: p<0.001 (t-test).

FIGS. 2A to 2D illustrate silver with ebselen in combination exhibitedsynergistic bactericidal effect. E. coli DHB4 grown to OD_(600 nm) of0.4 were treated with serial dilutions of ebselen and AgNO3 incombination. FIG. 2A is a line chart showing cell viability wasrepresented by measuring OD_(600 nm). The growth curves showed asynergistic bacteriostatic effect of Ag+ with ebselen in combination inLB medium. Five μM Ag+ and 40 μM ebselen in combination inhibited E.coli growth 480 min post-treatment (p=0.0075<0.01). FIG. 2B is a linechart showing changes of colony forming units of E. coli DHB4 on LBplates 0, 10, 60, 120, and 240 min post-treatment. The synergisticbactericidal effect of 5 μM Ag+ and 80 μM ebselen in combination wasconfirmed by the colony formation assay on LB-agar plates. Five μM Ag+and 80 μM ebselen in combination killed majority E. coli 60 minpost-treatment (p=0.00021<0.001). FIG. 2C is a group of FACS plots ofpropidium iodide (PI) stained E. coli DHB4, and FIG. 2D is a bar chartshowing mean values±s. d. (D) of PI stained E. coli DHB4. Five μM Ag+and 20 μM ebselen in combination enhanced the frequency of propidiumiodide (PI) staining (p=0.00083<0.001). In FIGS. 2A to 2C, data arepresented as means±s. d. of three independent experiments. *: p<0.05,**: p<0.01, ***: p<0.001 (t-test).

FIGS. 3A to 3F illustrate silver with ebselen in combination directlydisrupted bacterial Trx and GSH systems. E. coli DHB4 grown toOD_(600 nm) of 0.4 were treated with serial dilutions of ebselen andAgNO3 in combination. FIG. 3A is a bar chart showing TrxR activitieswere assayed using DTNB reduction in the presence of Trx in E. coliextracts, 50 mM Tris·HCl (pH 7.5), 200 μM NADPH, 1 mM EDTA, 1 mM DTNB,in the presence of 100 nM E. coli TrxR. Five μM Ag⁺ and 20 μM ebselen incombination resulted in a dramatic loss of TrxR activities(p=0.00018<0.001). FIG. 3B is a bar chart showing Trx activities wereassayed using DTNB reduction in the presence of Trx in E. coli extracts,50 mM Tris·HCl (pH 7.5), 200 μM NADPH, 1 mM EDTA, 1 mM DTNB, 5 μM E.coli Trx. Five μM Ag⁺ and 20 μM ebselen in combination resulted in adramatic loss of Trx activities (p=0.0036<0.01). FIG. 3C is a bar chartshowing changes of Trx1 redox state in E. coli upon ebselen and AgNO3treatment. E. coli were precipitated in 5% TCA and alkylated with 15 mMAMS, and the percent of reduced Trx1 were analyzed by Western blot. FIG.3D is a group of Western blot images showing changes of Trx2 redox statein E. coli upon ebselen and AgNO3 treatment. E. coli were precipitatedin 5% TCA and alkylated with 15 mM AMS, diamide oxidized Trx2 was usedas a Trx2 positive control, and the percent of reduced Trx2 wereanalyzed by Western blot. FIG. 3E is a bar chart showing GSH amountswere measured by GR-coupled DTNB reduction assay in E. coli extracts, 50mM Tris·HCl (pH 7.5), 200 μM NADPH, 1 mM EDTA, 1 mM DTNB, 50 nM GR. FiveμM Ag⁺ and 20 μM ebselen in combination treatment depleted thefunctional GSH in 10 min compared with control (p=0.000021<0.001). FIG.3F is a Western blot image showing changes of proteinsS-glutathionylation in E. coli. Cells were cultured, washed, andre-suspended in lysis buffer containing 30 mM IAM. After lysed bysonication, Western blotting assay was performed with IgG2a mousemonoclonal antibody (VIROGEN, 101-A/D8) for glutathione-proteincomplexes. In FIGS. 3A, 3B, and 3E, data are presented as means±s. d. ofthree independent experiments. *: p<0.05, **: p<0.01, ***: p<0.001(t-test).

FIGS. 4A to 4D illustrate inhibitory effects of silver on E. coli Trxsystem in vitro. FIG. 4A is a line chart showing inhibition of E. coliTrxR by AgNO3. Pure recombinant 100 nM TrxR, and 5 μM Trx mixture wereincubated with AgNO3 solution in the presence of 200 μM NADPH, and thentheir activities were detected by DTNB reduction assay. FIG. 4B is afluorescent spectra of a complex between reduced E. coli 10 μM Trx1 withAgNO3. Reduced 10 μM E. coli Trx1 protein was incubated with a serialconcentration of AgNO3 solution and the fluorescent spectra was detectedwith an excitation wavelength at 280 nm. Oxidized Trx1 (Trx-S₂) was usedas a control. FIG. 4C is a bar chart showing inhibition of Trx by AgNO3.After the treatment described in (B), Trx activity was assayed by a DTNBmethod in the presence of E. coli Trx1. FIG. 4D is a bar chart showinginhibition reversibility of E. coli Trx1 by AgNO3. Silver-inhibited E.coli Trx1 was passed through a desalting column to remove smallmolecules and then Trx activity was measured. E. coli Trx1 without theinhibition was used as a control. The inhibition of Trx1 by Ag⁺ wasirreversible since the Trx1 activity was not recovered after desalting(p=0.00021<0.001). Data are presented as means±s. d. of threeindependent experiments. *: p<0.05, **: p<0.01, ***: p<0.001 (t-test).

FIGS. 5A to 5C illustrate ROS was a determining factor for synergisticbactericidal effect of silver and ebselen. FIG. 5A shows FACS histogramsof H₂DCF-DA-stained E. coli. FIG. 5B is a bar chart of mean MFI±s. d. ofH₂DCF-DA-stained E. coli. E. coli DHB4 grown to OD_(600 nm) of 0.4 weretreated by 20 μM ebselen and 5 μM AgNO3. ROS level was detected by flowcytometry (CyAn adp, Beckman coulter). Treatment with either 5 μM Ag⁺ or20 μM ebselen alone did not change ROS concentrations, while thecombination of 5 μM Ag⁺ and 20 ebselen resulted in increased levels ofROS (p=0.00012<0.001). FIG. 5C is a bar chart showing detection of H₂O₂using the Amplex® Red Hydrogen Peroxide/Peroxidase Assay Kit(Invitrogen). Reactions containing 50 μM Amplex® Red reagent, 0.1 U/mLHRP and the indicated amount of H₂O₂ in 50 mM sodium phosphate buffer,pH 7.4, were incubated for 30 minutes at room temperature and detectedwith absorbance at 560 nm. Background determined for a non-H₂O₂ controlreaction, has been subtracted from each value. The enhanced H₂O₂generated by 5 μM Ag⁺ and 20 μM ebselen treated E. coli DHB4 cells wereverified (p=0.00057<0.0001). In FIGS. 5B and 5C, data are presented asmeans±s. d. of three independent experiments. *: p<0.05, **: p<0.01,***: p<0.001 (t-test).

FIGS. 6A to 6B illustrate mode of action of silver and ebselen in invivo mild and acute mice peritonitis model. FIG. 6A is a line chart ofE. coli CFU measurements over 36 hours in the mild mice peritonitismodel. Mice were infected by intraperitoneal administration of 100 μl1.7×10⁶ E. coli ZY-1 cells. After 24 h, 12 mice per group receivedantibacterial treatments (25 mg ebselen/kg and 6 mg AgNO₃/kg bodyweight). 12, 24, and 36 h after treatment, the peritoneal fluid wascollected for analysis of E. coli CFU (n=12 mice for each group)(p=0.0055<0.01), and data are presented as means±s. d. of threeindependent experiments. *: p<0.05, **: p<0.01, ***: p<0.001 (t-test).FIG. 6B is a line chart of E. coli CFU measurements over 96 hours in theacute mice peritonitis model. Inoculation was performed byintraperitoneal injection of 100 μl of 6.0×10⁶ CFU/ml E. coli ZY-1inoculums. After inoculation for 1 h, 10 mice per group receivedantibacterial treatments, and the mice were observed for 7 days toevaluate overall survival (n=10 mice for each group), and the experimentwas performed duplicate.

FIG. 7 is a chart showing that the Bliss model for synergy confirms asynergistic effect, between Ag+ and 4 antibiotics, against a modelGram-negative bacteria, E. coli. The degree of synergy was quantified,using the Bliss Model for Synergy, after 1 and 4 h of treatment with 5μM AgNO₃ in combination with the following antibiotics: 80 μMgentamicin, 80 μM kanamycin, 80 μM geneticin, 80 μM tetracycline, and 80μM Ebselen was used as positive control.

FIGS. 8A and 8B illustrate that ROS was a determining factor forsynergistic bactericidal effects of silver and antibiotics incombinations. E. coli DHB4 grown to OD_(600 nm) of 0.4 were treated with80 μM antibiotics and 5 μM AgNO₃ in combinations, and silver and ebselenin combination was used as a positive control. FIG. 8A is a bar chartshowing that ROS level was detected by flow cytometry (CyAnadp, Beckmancoulter), and mean MFI±. s. d. of H₂DCF-DA-stained E. coli weredetected. FIG. 8B is a bar chart showing that detection of H2O2 usingthe Amplex® Red Hydrogen Peroxide/Peroxidase Assay Kit (Invitrogen).Reactions containing 50 μM Amplex® Red reagent, 0.1 U/mL HRP and theindicated amount of H₂O₂ in 50 mM sodium phosphate buffer, pH 7.4, wereincubated for 30 minutes at room temperature and detected withabsorbance at 560 nm. Background determined by a non-H₂O₂ controlreaction has been subtracted from each value. Data are presented asmeans±s. d. of three independent experiments. *: p<0.05, **: p<0.01,***: p<0.001 (t-test).

FIGS. 9A to 9C illustrate that antibiotics alone could not directlydisrupt bacterial Trx system. E. coli DHB4 grown to OD_(600 nm) of 0.4were treated with antibiotics and AgNO3 in combinations for 10 min, andebselen and AgNO₃ in combination was used as a positive control. FIG. 9Ais a bar chart showing Trx activities assayed using DTNB reduction inthe presence of Trx in E. coli extracts. FIG. 9B is a bar chart showingTrxR activities assayed using DTNB reduction in the presence of TrxR inE. coli extracts. FIG. 9C is a Western blot image showing the percent ofreduced Trx1. E. coli DHB4 grown to OD_(600 nm) of 0.4 were treated withantibiotics and AgNO3 in combinations for 60 min, and ebselen and AgNO₃in combination was used as positive control. E. coli extracts wereprecipitated in 5% TCA and alkylated with 15 mM AMS and the percent ofreduced Trx1 was analyzed by Western blot. The mean±s. d. of threeindependent experiments was depicted. The t-test significances werecalculated between control and rest groups, and *: p<0.05, **: p<0.01,***: p<0.001.

FIGS. 10A and 10B illustrate that silver and conventional antibiotics incombinations could not directly disrupt the bacterial GSH system for 10minutes. E. coli DHB4 grown to OD_(600 nm) of 0.4 were treated withantibiotics and AgNO₃ in combinations for 10 min, and ebselen and AgNO₃in combination was used as a positive control. FIG. 10A is a bar chartshowing total GSH amounts measured by GR-coupled DTNB reduction assay inE. coli extracts. FIG. 10B is a Western blot image showing changes inprotein S-glutathionylation in E. coli. The mean s. d. of threeindependent experiments is depicted. The t-test significances werecalculated between control and test groups, and *: p<0.05, **: p<0.01,***: p<0.001.

FIGS. 11A and 11B illustrate that silver and conventional antibiotics incombinations could not directly disrupt the bacterial GSH system for 60minutes. E. coli DHB4 grown to OD_(600 nm) of 0.4 were treated withantibiotics and AgNO₃ in combinations for 60 min, and ebselen and AgNO₃in combination was used as a positive control. FIG. 11A is a bar chartshowing total GSH amounts were measured by GR-coupled DTNB reductionassay in E. coli extracts. FIG. 11B is a Western blot image showingchanges in protein S-glutathionylation in E. coli. The mean±s. d. ofthree independent experiments was depicted. The t-test significanceswere calculated between control and rest groups, and *: p<0.05, **:p<0.01, ***: p<0.001.

DETAILED DESCRIPTION

The disclosure herein is generally directed to antibiotic compositionscomprising multiple pharmaceutically active agents (two, three, four, ormore) that are useful in combination as antimicrobial therapeutics thattreat and/or prevent bacterial infection by killing or inhibiting thegrowth of bacteria. For example, a composition that can comprise ametal-containing agent (e.g., silver ion) and an antimicrobial agent(e.g., ebselen) in synergistic combination disclosed herein targetsbacterial thioredoxin and glutathione systems and is potent againstbacterial infections such as those caused by Gram-negative bacteria.

An “effective amount” when used in connection with a composition oractive agent disclosed herein is an amount sufficient to produce atherapeutic result in a subject in need thereof. For example atherapeutic result includes, but is not limited to, treating,preventing, ameliorating, or lessening bacterial infection and/or anysymptom thereof such as inflammation, fever, cough, sneezing, nasalcongestion, runny nose, sore throat, pain, nausea, vomiting, orconstipation in a subject.

The term “about” means the referenced numeric indication plus or minus15% of that referenced numeric indication.

The present disclosure shows that multidrug-resistant (MDR)Gram-negative bacteria are highly sensitive to silver and ebselen in asynergistic combination. In contrast, silver shows no synergistictoxicity with ebselen against mammalian cells. Biochemical experimentsrevealed that silver and ebselen caused a fast depletion of glutathioneand inhibition of the thioredoxin system in bacteria. Silver ions wereidentified as strong inhibitors of E. coli thioredoxin and thioredoxinreductase, which are required for ribonucleotide reductase and DNAsynthesis and defense against oxidative stress. Bactericidal efficacy ofsilver and ebselen causing oxidative stress was further verified in thetreatment of mild and acute MDR E. coli peritonitis in mice. Theseresults demonstrate that thiol-dependent redox systems in bacteria couldbe targeted in the design of new antibacterial drugs. Silver and ebselenact as a probe to target essential bacterial systems which might bedeveloped for novel efficient treatments against MDR Gram-negativebacterial infections. Silver acted strongly synergistic with theselenazol drug ebselen, to combat difficult-to-treat MDR Gram-negativebacteria in the clinic, by targeting thiol-dependent antioxidantsystems. The results were further proven by successfully treating micewith MDR E. coli caused mild or acute peritonitis. Redox system is auniversal anti-oxidative system which is essential for living organism,inhibition of redox system will result in oxidative stress, which showsa novel antibacterial principle to screen and use new antibiotics.

In some cases, an antibiotic composition disclosed herein kills MDRGram-negative bacteria. In some instances, the antibiotic compositionselectively targets bacterial thiol-dependent redox systems via strongbactericidal effect of silver and ebselen in synergistic combinationagainst GSH-positive bacterial infections, particularly on MDRGram-negative bacteria. In some instances, the silver ions are stronginhibitors of both E. coli Trx and TrxR, and the combination withebselen depletes GSH and gave a steep rise in ROS generation. In someinstances, the presence of ebselen improves efficacy of silver and thusdecreases the antibacterial concentration of silver needed to elicit aneffect, with highly significant selective toxicity on bacteria overmammalian cells. This selective toxicity facilitates the systemicmedical application of silver in the treatment of MDR Gram-negativebacteria. In some instances, the synergistic bactericidal effect of Ag⁺with ebselen in combination is efficient against these MDR Gram-negativepathogens (Table 1). Further, results from animal experiments indicatedthat this antibiotic combination can be considered as a candidate forclinical trials against MDR bacteria (FIGS. 6A-6B, Table 1). Silver andebselen together can be regarded as a probe targeting essentialfunctions in bacteria. The experimental results presented here proposedmechanisms for the synergistic antibacterial effect of Ag⁺ with ebselenin combination. Silver and ebselen can directly inhibit E. coli TrxR,and fast deplete GSH, which resulted in the elevation of ROS productionto determine cell death (synopsis). Thiol-dependent redox pathwaysregulate various central cellular functions. Thus, Ag⁺ with ebselen incombination can react with SH-groups in GSH, and particularly Trx andTrxR and possibly many other proteins, indicating that the inhibitoryeffect of Ag⁺ with ebselen in combination may involve several cellulartargets. In addition, Ag⁺ and ebselen might target other molecules: forexample, diguanylate cyclase and M. tuberculosis antigen 85. This mayimpair the development of antibiotic resistance in bacteria.

Active Agents

Disclosed herein is a combination of a metal-containing agent and anantimicrobial agent. A metal-containing agent can comprise a metal ormetal ion disclosed herein. The metal-containing agent can comprise ametal ion that possesses antibiotic activity, for example silver,copper, zinc, mercury, tin, lead, bismuth, cadmium, cerium, chromium,and thallium ions. Antimicrobial metal ions of silver, gold, copper andzinc, can be considered safe for in vivo use and not substantiallyabsorbed into the body. The antimicrobial agent can be an antibioticsuch as gentamicin, kanamycine, geneticin, tetracycline, a nonoragoselenum agent, or an organoselenium agent (e.g., ebselen or ananalog thereof), or any antimicrobial agent described herein. In someinstances, a metal-containing agent (e.g., silver) enhances theantibacterial effects of an organoselenium agent (e.g., ebselen) orcertain antibiotics against Gram-negative bacteria through directtargeting the bacterial thioredoxin (Trx) system, the glutathione (GSH)system, or both. In some instances, targeting/attacking the GSH systemincreases efficacy of an antibiotic composition in killing one or morebacteria. In some instances, an antibiotic composition can comprise asilver agent and an antibiotic disclosed herein.

In some cases, an antibiotic composition disclosed herein can comprise ametal containing agent, either in the form of a metal atom or a metalion unlinked or linked to another molecule via a covalent or noncovalent(e.g., ionic) linkage. Silver containing agents can include but are notlimited to covalent compounds such as silver dihydrogen citrate, silversulfadiazine and silver salts such as silver oxide, silver carbonate,silver deoxycholate, silver salicylate, silver iodide, silver nitrate,silver paraaminobenzoate, silver paraaminosalicylate, silveracetylsalicylate, silver ethylenediaminetetraacetic acid (“Ag EDTA”),silver picrate, silver protein, silver citrate, silver lactate andsilver laurate. The silver agents can be covalent compounds or forexample, silver salts, silver complex ions, colloidal silver,silver/zeolite composites, silver/phosphate, silver/glass particles(antimicrobial, controlled release), or any mixture thereof. In someinstances, silver salts are silver chloride, silver nitrate, silveracetate, silver benzoate, silver bromate, silver chlorate, silverlactate, silver molybdate, silver nitrite, silver(I) oxide, silverperchlorate, silver permanganate, silver selenate, silver selenite,silver sulfadiazine, silver sulfate, and mixtures thereof. In someinstances, silver complex ions are silver chloro complex ions, silverthiosulfato complex ions, or mixtures thereof. In some instances,colloidal silver particles are silver nanoparticles.

In some instances, the metal containing agent can comprise a metal salt.The metal salt can be a silver salt as silver nitrate, silver acetate,silver benzoate, silver carbonate, silver iodate, silver iodide, silverlactate, silver laurate, silver oxide, silver palmitate, silver protein,or silver sulfadiazine. The metal containing agent can comprise a copperion source such as copper(II) nitrate, copper sulfate, copperperchlorate, copper acetate, tetracyan copper potassium. The metalcontaining agent can comprise a zinc ion source such as zinc(II)nitrate, zinc sulfate, zinc perchlorate, zinc acetate and zincthiocyanate; such a mercury ion source as mercury perchlorate, mercurynitrate and mercury acetate. The metal containing agent can comprise atin ion source such as tin sulfate. The metal containing agent cancomprise a lead ion source such as lead sulfate and lead nitrate. Themetal containing agent can comprise a bismuth ion source such as bismuthchloride and bismuth iodide. The metal containing agent can comprise acadmium ion source such as cadmium perchlorate, cadmium sulfate, cadmiumnitrate and cadmium acetate. The metal containing agent can comprise achromium ion source such as chromium perchlorate, chromium sulfate,chromium ammonium sulfate and chromium acetate. The metal containingagent can comprise a thallium ion source such as thallium ion source asthallium perchlorate, thallium sulfate, thallium nitrate or thalliumacetate. The silver may be provided in a soluble or insoluble form, suchas silver chloride, adsorbed on a support or particles selected from thegroup consisting of titanium oxide, magnesium oxide, aluminum oxide,silicon oxide, calcium oxide, barium oxide, calcium hydroxyapatite,chalk, natural ground or precipitated calcium carbonates, calciummagnesium carbonates, silicates, sheet silicates, zeolites, clays,bentonites and titanium oxide. Insoluble silver on a support materialcan be useful for topical application. The composition disclosed hereinmay also include an effective amount of a dispersant, such aspolynaphthalenesulfonate, naphthalenesulfonate or alkyl sulfosuccinate.

In some instances, the minimal inhibitory concentration (MIC) to one ormore bacteria for an antibiotic metal (e.g., silver) contained in anantibiotic composition disclosed herein can be less than about: 50 μM,25 μM, 20 μM, 10 μM, 5 μM, 1 μM, 0.5 μM, 0.1 μM, 50 nM, 25 nM, 20 nM, 10nM, 5 nM, or 1 nM.

Organoselenium agents are chemical compounds containingcarbon-to-selenium chemical bonds. Selenium can exist with oxidationstate −2, +2, +4, +6, e.g., Se (II). Organoselenium agents include butare not limited to selenols, diselenides, selenyl halides, selenides(selenoethers), selenoxides, selenones, selenenic acids, seleninicacids, perseleninic acids, selenuranes, seleniranes, selones (e.g.,selenourea), selenocysteine, selenomethionine, diphenyldiselenide,benzeneselenol. In some instances, the organoselenium agent can compriseselenazol or isoselenazol compound, for example abenzoisoselenazol-3(2H)-one compound, e.g., ebselen (Chemical name:2-phenyl-1,2-benzisoselenazol-3(2H)-one, IUPAC name:2-Phenyl-1,2-benzoselenazol-3-one), ebselen diselenide, or a structuralanalog such as those disclosed herein.

In some instances, the organoselenium agent can comprise a compoundrepresented by the following general formula (I) or (I′):

wherein R¹ and R² independently represent a hydrogen atom, a halogenatom, a trifluoromethyl group and the like; R³ represents an aryl group,an aromatic heterocyclic group and the like; R⁴ represents a hydrogenatom, a hydroxyl group, a —S-α-amino acid group and the like; R⁵represents a hydrogen atom or a C₁-C₆ alkyl group; Y represents oxygenatom or sulfur atom; n represents an integer of from 0 to 5; and theselenium atom may be oxidized, whose example includes2-phenyl-1,2-benzisoselenazol-3(2H)-one or a ring-opened form thereof.In some instances, the organoselenium agent can comprise a compoundselected from the group consisting of2-phenyl-1,2-benziso-selenazol-3(2H)-one or a ring-opened form thereofand a physiologically acceptable salt thereof. In some instances, theorganoselenium agent can comprise a substance selected from the groupconsisting of 2-phenyl-1,2-benziso-selenazol-3(2H)-one or a ring-openedform thereof and a physiologically acceptable salt thereof. In someinstances, the organoselenium agent can comprise a substance selectedfrom the group consisting of 2-phenyl-1,2-benziso-selenazol-3(2H)-one ora ring-opened form thereof and a physiologically acceptable saltthereof.

As the C₁-C₆ alkyl group represented by R¹ and R², either a straight ora branched chain alkyl group may be used, and examples include methylgroup, ethyl group, n-propyl group, isopropyl group, cyclopropyl group,n-butyl group, sec-butyl group, isobutyl group, tert-butyl group,n-pentyl group, and n-hexyl group. As the C₁-C₆ alkoxyl grouprepresented by R¹ and R², either a straight or a branched chain alkoxylgroup may be used, and examples include methoxy group, ethoxy group,n-propoxy group, isopropoxy group, n-butoxy group, sec-butoxy group,tert-butoxy group, n-pentoxy group, and n-hexoxy group.

As the aryl group represented by R³, for example, a monocyclic to atricyclic, preferably a monocyclic or a bicyclic aryl group having 6 to14 carbon atoms, preferably 6 to 10 carbon atoms can be used. Morespecifically, phenyl group or naphthyl group and the like are preferred.As the aromatic heterocyclic group represented by R³, for example, amonocyclic to a tricyclic, preferably a monocyclic or a bicyclicaromatic heterocyclic group containing one ore more heteroatoms such asnitrogen atom, oxygen atom and sulfur atom can be used. When two or moreheteroatoms are contained, they may be same or different. Examplesinclude thienyl group, furyl group, pyrrolyl group, imidazolyl group,pyrazolyl group, isoxazolyl group, pyridyl group, pyrazinyl group,pyrimidinyl group, pyridazinyl group, indolizinyl group, isoindolylgroup, indolyl group, isoquinolyl group, quinolyl group, phthalazinylgroup, naphthylidinyl group, quinoxalinyl group, quinazolinyl group,cinnolinyl group, pteridinyl group, carbazolyl group, acridinyl group,phenanthridinyl group, and phenothiazinyl group.

The aryl group, the aromatic heterocyclic group, the 5- to 7-memberedcycloalkyl group, or the 5- to 7-membered cycloalkenyl group representedby R³ may have one or more substituents on the ring. When the ring issubstituted with two or more substituents, they may be same ordifferent. The position of the substituent is not particularly limited,and the substituent may be present at any position on the ring. The typeof the substituent is not particularly limited, and examples include aC₁-C₆ alkyl group, a C₂-C₆ alkenyl group, a C₂-C₆ alkynyl group, aC₆-C₁₄ aryl group, a heterocyclic group (the heterocycle used hereinincludes aromatic heterocyclic groups and partially saturated orsaturated heterocyclic groups), a halogen atom (the halogen atom usedherein may be any one of fluorine atom, chlorine atom, bromine atom, oriodine atom), hydroxyl group, oxo group, amino group, ammonium group,imino group, mercapto group, thioxo group, cyano group, nitro group,carboxyl group, phosphate group, sulfo group, hydrazino group, a C₁-C₆ureido group, a C₁-C₆ imido group, isothiocyanate group, isocyanategroup, a C₁-C₆ alkoxyl group, a C₁-C₆ alkylthio group, a C₆-C₁₄ aryloxygroup, a heterocyclic-oxy group, a C₆-C₁₄ arylthio group, aheterocyclic-thio group, a C₇-C₁₅ aralkyl group, a heterocycle-alkylgroup, a C₇-C₁₅ aralkyloxy group, a heterocyclic-alkyloxy group, a C₁-C₆alkoxycarbonyl group, a C₆-C₁₄ aryloxycarbonyl group, aheterocyclic-oxycarbonyl group, a C₂-C₇alkylcarbonyl group, a C₆-C₁₄arylcarbonyl group, a heterocyclic-carbonyl group, aC₂-C₇alkylcarbonyloxy group, a C₆-C₁₄ arylcarbonyloxy group, aheterocyclic-carbonyl oxygroup, a C₂-C₈ alkylcarbonylamino group, aC₁-C₆ sulfonyl group, a C₁-C₆ sulfinyl group, a C₁-C₆ sulfonylaminogroup, a C₁-C₆ carbamoyl group, and a C₂-C₆ sulfamoyl group.

The substituents exemplified above may be further substituted with oneor more other substituents. Examples of such substituents include ahydroxy-C₁-C₆ alkyl group, a halogenated-C₁-C₆ alkyl group, a mono- ordi-C₁-C₆ alkylamino group, a halogenated-C₁-C₆ alkylcarbonyl group, ahalogenated-C₆-C₁₄ aryl group, a hydroxy-C₆-C₁₄ aryl group, and a mono-or di-C₁-C₆ alkylcarbamoyl group. However, the substituents explainedabove are referred to only for exemplification, and the substituentsused are not limited to these examples.

Although the type of the —S-α-amino acid group represented by R⁴ is notparticularly limited, the group may preferably be an amino acid residuecontaining thiol group. The —S-α-amino acid residue may be a residue ofan amino acid which constitutes a protein or a peptide compound. Thetype of proteins or peptide compounds is not particularly limited so faras they are physiologically acceptable. For example, serum protein suchas albumin and globulin may preferably be used. Among serum protein,albumin is more preferred, and human albumin is particularly preferred.Examples of the aralkyl group represented by R⁴ whose aryl moiety mayoptionally be substituted with one or more substituents include benzylgroup, parahydroxybenzyl group, and 2,4-dihydrobenzyl group. R⁴ and R⁵may combine together to represent single bond, and in that case, a5-membered ring is formed which contains the nitrogen atom bound to R⁵and the selenium atom. As the C₁-C₆ alkyl group represented by R⁵, thoseexemplified above can be used.

Physiologically acceptable salts of the compounds represented by theaforementioned general formula (I) or (I′) may be used. Thephysiologically acceptable salt can suitably be chosen by the personskilled in the art. Hydrates of the compounds as free form orphysiologically acceptable salts may also be used. When the compoundrepresented by the aforementioned general formula (I) or (I′) has one ormore asymmetric carbon atoms, stereoisomers such as optical isomers anddiastereoisomers, any mixture of the stereoisomers, racemates and thelike may be used.

In some instances, the organoselenium agent can comprise an ebselen oran analog thereof, such as a compound having a formula of:

or a pharmaceutically acceptable salt thereof,wherein X is selenium or sulfur, andwherein R is selected from the group consisting of: H, alkyl having acarbon chain of 1 to 14 carbon atoms wherein the carbon chain isbranched or unbranched which is optionally substituted withbensisoselenazol-3(2H)-one-2-yl, bensisotiazol-3(2H)-one-2-yl, OH,alkoxyl, SH, NH₂, N-alkylamino, N,N-dialkylamino, COOH, aryl which isoptionally substituted with C₁-C₅ alkyl, OH, alkoxyl, SH, NH₂,N-alkylamino, N,N-dialkylamino, COOH, CHO, NO₂, F, Cl, Br, I, andheteroaryl which is optionally substituted with C₁-C₅ alkyl, OH,alkoxyl, SH, NH₂, N-alkylamino, N,N-dialkylamino, COOH, CHO, NO₂, F, Cl,Br, and I, aryl which is optionally substituted with C₁-C₅ alkyl, OH,alkoxyl, SH, NH₂, N-alkylamino, N,N-dialkylamino, COOH, CHO, NO₂, F, Cl,Br, and I, heteroaryl which is optionally substituted with C₁-C₅ alkyl,OH, alkoxyl, SH, NH₂, N-alkylamino, N,N-dialkylamino, COOH, CHO, NO₂, F,Cl, Br, and I, and wherein A represents a saturated, unsaturated orpolyunsaturated 3 to 6 member carbon chain wherein N may optionallysubstitute for one or more carbons, and which is optionally substitutedwith one or more of OR, SR, and alkylamino, C₁-C₅ alkyl, OH, alkoxyl,SH, NH₂, N-alkylamino, N,N-dialkylamino, COOH, CHO, NO₂, F, Cl, Br, andI.

In some instances, the organoselenium agent can comprise an ebselenhaving a chemical structure of:

In some instances, the organoselenium agent can comprise an ebselenstructural analog, such as from classes of benzisoselenazol-3(2H)-one-aryl,-alkyl, 2-pyridyl or 4-pyridyl substitutedbenzisoselenazol-3 (2H)-ones, bisbenzisoselenazol-3 (2H)-ones,7-azabenzisoselenazol-3(2H)-one, selenamide, and bis(2-carbamoyl)phenyldiselenide, e.g., having a chemical structure below.

In some instances, the minimal inhibitory concentration (MIC) to one ormore bacteria for an organoselenium agent (e.g., ebselen) contained inan antibiotic composition disclosed herein can be less than about: 100μM, 90 μM, 80 μM, 70 μM, 60 μM, 50 μM, 40 μM, 30 μM, 25 μM, 20 μM, 15μM, 10 μM, 5 μM, 1 μM, 0.5 μM, or 0.1 μM.

Methods and compositions presented herein can utilize an active agent ina freebase, salt, hydrate, polymorph, isomer, diastereomer, prodrug,metabolite, ion pair complex, or chelate form. An active agent can beformed using a pharmaceutically acceptable non-toxic acid or base,including an inorganic acid or base, or an organic acid or base. In someinstances, an active agent that can be utilized in connection with themethods and compositions presented herein can be a pharmaceuticallyacceptable salt derived from acids including, but not limited to, thefollowing: acetic, alginic, anthranilic, benzenesulfonic, benzoic,camphorsulfonic, citric, ethenesulfonic, formic, fumaric, furoic,galacturonic, gluconic, glucuronic, glutamic, glycolic, hydrobromic,hydrochloric, isethionic, lactic, maleic, malic, mandelic,methanesulfonic, mucic, nitric, pamoic, pantothenic, phenylacetic,phosphoric, propionic, salicylic, stearic, succinic, sulfanilic,sulfuric, tartaric acid, or p-toluenesulfonic acid. In some instances,the active agent can be a salt of methanesulfonic acid.

In some instances, an antibiotic composition disclosed herein canfurther comprise or can be co-administered with one or moreantibacterial/antimicrobial drugs, for example amikacin, azithromycin,cefixime, cefoperazone, cefotaxime, ceftazidime, ceftizoxime,ceftriaxone, chloramphenicol, ciprofloxacin, clindamycin, colistin,domeclocycline, doxycycline, erythromycin, gentamicin, mafenide,methacycline, minocycline, neomycin, norfloxacin, ofloxacin,oxytetracycline, polymyxin B, pyrimethamine, sulfacetamide,sulfisoxazole, tetracycline, tobramycin, trimethoprim, or anycombination thereof.

In some instances, an antibiotic composition disclosed herein canfurther comprise or can be co-administered with oxazolidinoneantibacterial drug(s), and/or one or more drug(s) selected fromacebutolol, aceclidine, acetylsalicylic acid, N4 acetylsulfisoxazole,alclofenac, alprenolol, amfenac, amiloride, aminocaproic acid,aminoclonidine, aminozolamide, anisindione, apafant, atenolol,bacitracin, benoxaprofen, benoxinate, benzofenac, bepafant,betamethasone, betaxolol, bethanechol, brimonidine, bromfenac,bromhexine, bucloxic acid, bupivacaine, butibufen, carbachol, carprofen,celecoxib, cephalexin, chloramphenicol, chlordiazepoxide, chlorprocaine,chlorpropamide, chlortetracycline, cicloprofen, cinmetacin,ciprofloxacin, clidanac, clindamycin, clonidine, clonixin, clopirac,cocaine, cromolyn, cyclopentolate, cyproheptadine, demecarium,dexamethasone, dibucaine, diclofenac, diflusinal, dipivefrin,dorzolamide, enoxacin, epinephrine, erythromycin, eserine, estradiol,ethacrynic acid, etidocaine, etodolac, fenbufen, fenclofenac, fenclorac,fenoprofen, fentiazac, flufenamic acid, flufenisal, flunoxaprofen,fluorocinolone, fluorometholone, flurbiprofen and esters thereof,fluticasone propionate, furaprofen, furobufen, furofenac, furosemide,gancyclovir, gentamicin, gramicidin, hexylcaine, homatropine,hydrocortisone, ibufenac, ibuprofen and esters thereof, idoxuridine,indomethacin, indoprofen, interferons, isobutylmethylxanthine,isofluorophate, isoproterenol, isoxepac, ketoprofen, ketorolac,labetolol, lactorolac, latanoprost, levo-bunolol, lidocaine, lonazolac,loteprednol, meclofenamate, medrysone, mefenamic acid, mepivacaine,metaproterenol, methanamine, methylprednisolone, metiazinic, metoprolol,metronidazole, minopafant, miroprofen, MK-663, modipafant, nabumetome,nadolol, namoxyrate, naphazoline, naproxen and esters thereof, neomycin,nepafenac, nitroglycerin, norepinephrine, norfloxacin, nupafant,olfloxacin, olopatadine, oxaprozin, oxepinac, oxyphenbutazone,oxyprenolol, oxytetracycline, parecoxib, penicillins, perfloxacin,phenacetin, phenazopyridine, pheniramine, phenylbutazone, phenylephrine,phenylpropanolamine, phospholine, pilocarpine, pindolol, pirazolac,piroxicam, pirprofen, polymyxin, polymyxin B, prednisolone, prilocaine,probenecid, procaine, proparacaine, protizinic acid, rimexolone,rofecoxib, salbutamol, scopolamine, sotalol, sulfacetamide, sulfanilicacid, sulindac, suprofen, tenoxicam, terbutaline, tetracaine,tetracycline, theophyllamine, timolol, tobramycin, tolmetin,triamcinolone, trimethoprim, trospectomycin, valdecoxib, vancomycin,vidarabine, vitamin A, warfarin, zomepirac, and pharmaceuticallyacceptable salts thereof.

Formulations

In some cases, the present disclosure provides an antibioticcomposition, wherein the antibiotic composition comprises: asilver-containing agent; and an organoselenium agent. In some instances,the silver-containing agent can comprise a silver ion. In someinstances, the silver-containing agent can comprise silver nitrate. Insome instances, the silver-containing agent can comprise silverdihydrogen citrate. In some instances, the organoselenium agent cancomprise a selenazol compound. In some instances, the organoseleniumagent can comprise a benzoisoselenazol-3(2H)-one compound. In someinstances, the organoselenium agent can comprise an ebselen. In someinstances, the antibiotic composition can be in a dosage form of liquid.In some instances, the antibiotic composition can be in a dosage form ofa solution or a suspension. In some instances, a concentration of thesilver-containing agent in the antibiotic composition can be about 0.5to 50 μM, about 1 to 25 μM, or about 1 to 10 μM. In some instances, aconcentration of the silver-containing agent in the antibioticcomposition can be about 5 μM. In some instances, a concentration of theorganoselenium agent in the antibiotic composition can be about 4 to 25μM, about 30 to 200 μM, about 30 to 150 μM, or about 30 to 100 μM. Insome instances, a concentration of the organoselenium agent in theantibiotic composition can be about 40 μM or about 80 μM. In someinstances, the silver-containing agent and the organoselenium agent canbe a molar ratio of about 1:2 to about 1:20. In some instances, thesilver-containing agent and the organoselenium agent can be a molarratio of about 1:4, 1:8, or 1:16. In some instances, the antibioticcomposition exhibits an IC₅₀ value of about 10-100 nM to one or moreGram-negative bacteria or Gram-positive bacteria. In some instances, theantibiotic composition exhibits an IC₅₀ value of about 50 nM or lower toone or more Gram-negative bacteria or Gram-positive bacteria. In someinstances, the one or more Gram-negative bacteria can comprise K.pneumonia, A. baumannii, P. aeruginosa, E. cloacae, E. coli, or anycombination thereof. In some instances, the antibiotic composition cancomprise AgNO₃ and ebselen. In some instances, a composition disclosedherein can be in a liquid dosage form. In some instances, the antibioticcomposition can comprise 5 μM of AgNO₃ and 4 μM of ebselen in a liquiddosage form. In some instances, the antibiotic composition can comprise5 μM of AgNO₃ and 20 μM of ebselen in a liquid dosage form. In someinstances, the antibiotic composition can comprise 5 μM of AgNO₃ and 40μM of ebselen in a liquid dosage form. In some instances, the antibioticcomposition can comprise 5 μM of AgNO₃ and 80 μM of ebselen in a liquiddosage form.

In some cases, the present disclosure provides a pharmaceuticalformulation that can comprise the antibiotic composition disclosedherein. In some instances, the pharmaceutical formulation further cancomprise an excipient disclosed herein.

In some cases, the present disclosure provides a method of making anantibiotic composition, comprising mixing a silver-containing agent andan organoselenium agent. In some instances, the mixing can be conductedin a liquid disclosed herein, for example a suspension, a colloid, or asolution. In some instances, the liquid comprises one or more activeagents or excipients disclosed herein. In some instances, the mixing cancomprise adding the silver-containing agent to a liquid that cancomprise the organoselenium agent. In some instances, the mixing cancomprise adding the organoselenium agent to a liquid that can comprisethe silver-containing agent.

In some instances, an active agent disclosed herein can be present inabout: 0.01-0.1, 0.1-1, 1-10, 1-20, 5-30, 5-40, 5-50, 10-20, 10-25,10-30, 10-40, 10-50, 15-20, 15-25, 15-30, 15-40, 15-50, 20-30, 20-40,20-50, 20-100, 30-40, 30-50, 30-60, 30-70, 30-80, 30-90, 30-100, 40-50,40-60, 40-70, 40-80, 40-90, 40-100, 50-60, 50-70, 50-80, 50-90, 50-100,50-150, 50-200, 50-300, 100-300, 100-400, 100-500, 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350,400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or 1000 μM,or any combination thereof. In some instances, an active agent disclosedherein can be present in about: 1 mg-2.5 mg, 2.5-25 mg, 2.5-30 mg, 5-20mg, 5-15 mg, 5-10 mg, 10-15 mg, 10-20 mg, 10-25 mg, 11.5-13 mg, 5 mg,5.5 mg, 6 mg, 6.5 mg, 7 mg, 7.5 mg, 8 mg, 8.5 mg, 9 mg, 9.5 mg, 10 mg,10.5 mg, 11 mg, 11.5 mg, 12 mg, 12.5 mg, 13 mg, 13.5 mg, 14 mg, 14.5 mg,15 mg, 16 mg, 17 mg, 18 mg, 19 mg, or 20 mg. In some instances, anactive agent disclosed herein can be present in about: 5-50 mg, 5-40 mg,5-30 mg, 10-25 mg, 15-20 mg, 10 mg, 11 mg, 12 mg, 13 mg, 14 mg, 15 mg,16 mg, 17 mg, 18 mg, 19 mg, 20 mg, 21 mg, 22 mg, 23 mg, 24 mg, 25 mg, 30mg, 35 mg, 40 mg, 45 mg, or 50 mg, or any combination thereof. In someinstances, two active agents are present in a molar or weight ratio byweight of about: 1:10 to 1:30, 1:20 to 1:30, 1:10 to 1:20, 1:1 to 1:15,or 1:1 to 1:0, 1:1 to 1:5, 1:1 to 1:4, 1:1 to 1:3, or 1:1 to 1:2. Insome instances, two active agents are present in a molar or weight ratioby weight of about: 1:30, 1:29, 1:28, 1:27, 1:26, 1:25, 1:24, 1:23,1:22, 1:21, 1:20, 1:19, 1:18, 1:17, 1:16, 1:15, 1:14, 1:13, 1:12, 1:11,1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2, or 1:1.

In some cases, an antibiotic composition can comprise multiple activeagents administered of at least about 0.001 mg, for example, at leastabout: 0.01 mg, 0.1 mg, 0.2 mg, 0.3 mg, 0.4 mg, 0.5 mg, 0.6 mg, 0.7 mg,0.8 mg, 0.9 mg, 1 mg, 1.5 mg, 2 mg, 2.5 mg, 3 mg, 3.5 mg, 4 mg, 4.5 mg,5 mg, 5.5 mg, 6 mg, 6.5 mg, 7 mg, 7.5 mg, 8 mg, 8.5 mg, 9 mg, 9.5 mg, or10 mg, per kg body weight of a subject in need thereof. The powdercomposition may comprise a total dose of an active agent administered atabout 0.1 to about 10.0 mg, for example, about 0.1-10.0 mg, about0.1-9.0 mg, about 0.1-8.0 mg, about 0.1-7.0 mg, about 0.1-6.0 mg, about0.1-5.0 mg, about 0.1-4.0 mg, about 0.1-3.0 mg, about 0.1-2.0 mg, about0.1-1.0 mg, about 0.1-0.5 mg, about 0.2-10.0 mg, about 0.2-9.0 mg, about0.2-8.0 mg, about 0.2-7.0 mg, about 0.2-6.0 mg, about 0.2-5.0 mg, about0.2-4.0 mg, about 0.2-3.0 mg, about 0.2-2.0 mg, about 0.2-1.0 mg, about0.2-0.5 mg, about 0.5-10.0 mg, about 0.5-9.0 mg, about 0.5-8.0 mg, about0.5-7.0 mg, about 0.5-6.0 mg, about 0.5-5.0 mg, about 0.5-4.0 mg, about0.5-3.0 mg, about 0.5-2.0 mg, about 0.5-1.0 mg, about 1.0-10.0 mg, about1.0-5.0 mg, about 1.0-4.0 mg, about 1.0-3.0 mg, about 1.0-2.0 mg, about2.0-10.0 mg, about 2.0-9.0 mg, about 2.0-8.0 mg, about 2.0-7.0 mg, about2.0-6.0 mg, about 2.0-5.0 mg, about 2.0-4.0 mg, about 2.0-3.0 mg, about5.0-10.0 mg, about 5.0-9.0 mg, about 5.0-8.0 mg, about 5.0-7.0 mg, about5.0-6.0 mg, about 6.0-10.0 mg, about 6.0-9.0 mg, about 6.0-8.0 mg, about6.0-7.0 mg, about 7.0-10.0 mg, about 7.0-9.0 mg, about 7.0-8.0 mg, about8.0-10.0 mg, about 8.0-9.0 mg, or about 9.0-10.0 mg, per kg body weightof a subject in need thereof.

In some instances, a composition disclosed herein can comprise two ormore active agents (e.g., a metal compound, ebselen or a derivativethereof), each of which can be independently present at a dose of about:1-10 mg, 2.5-30 mg, 2.5-20 mg, 1-20 mg, 1-30 mg, 5-30 mg, 10-40 mg,20-50 mg, 30-60 mg, 40-70 mg, 50-80 mg, 60-90 mg, or 1-100 mg, includingbut not limited to about: 1.0 mg, 1.5 mg, 2.5 mg, 3.0 mg, 4.0 mg, 5.0mg, 6.0 mg, 6.5 mg, 7.0 mg, 7.5 mg, 8.0 mg, 8.5 mg, 9.0 mg, 9.5 mg,10.0, 10.5 mg, 11.0 mg, 12.0 mg, 12.5 mg, 13.0 mg, 13.5 mg, 14.0 mg,14.5 mg, 15.0 mg, 15.5 mg, 16 mg, 16.5 mg, 17 mg, 17.5 mg, 18 mg, 18.5mg, 19 mg, 19.5 mg, 20 mg, 20.5 mg, 21 mg, 21.5 mg, 22 mg, 22.5 mg, 23mg, 23.5 mg, 24 mg, 24.5 mg, 25 mg, 25.5 mg, 26 mg, 26.5 mg, 27 mg, 27.5mg, 28 mg, 28.5 mg, 29 mg, 29.5 mg, 30 mg, 30.5 mg, 31 mg, 31.5 mg, 32mg, 32.5 mg, 33 mg, 33.5 mg, 36 mg, 36.5 mg, 37 mg, 37.5 mg, 38 mg, 38.5mg, 39 mg, 39.5 mg, 40 mg, 40.5 mg, 41 mg, 41.5 mg, 42 mg, 42.5 mg, 43mg, 43.5 mg, 44 mg, 44.5 mg, 45 mg, 45.5 mg, 46 mg, 46.5 mg, 47 mg, 47.5mg, 48 mg, 48.5 mg, 49 mg, 49.5 mg, 50 mg, 55 mg, 60 mg, 65 mg, 70 mg,75 mg, 80 mg, 85 mg, 90 mg, 95 mg, or 100 mg.

Excipients

In some instances, the antibiotic composition disclosed herein canfurther comprise one or more excipients, e.g., different substance, orsame substance but different sizes. In some instances, the excipient cancomprise a carrier, e.g., water-insoluble polysaccharide oroligosaccharide. In some instances, the carrier can be selected from agroup consisting of cellulose acetate, cellulose acetate butyrate,cellulose acetate propionate, cellulose acetate phthalate, chitosan,β-cyclodextrin, ethyl cellulose, hydroxypropylmethyl cellulose phthalate(HPMCP), microcrystalline cellulose, starch, and any combinationthereof. In some instances, the excipient can comprise a thickeningagent, e.g., a water-soluble polysaccharide. In some instances, thethickening agent can be selected from the group consisting of hydroxypropyl methyl cellulose (HPMC), acacia, alginic acid, colloidal siliconedioxide, carboxymethylcellulose calcium, gelatin, hydroxy propylcellulose, hydroxyl propyl cellulose (hypromellose), methyl cellulose,sucrose, sodium alginate, sodium carboxy methyl cellulose, and anycombination thereof. In some instances, the excipient can comprise afirst excipient (any excipient disclosed herein) and a second excipient(any excipient disclosed herein). In some instances, the excipient cancomprise a carrier (e.g., microcrystalline cellulose) and a thickeningagent (e.g., HPMC).

In some instances, the antibiotic composition disclosed herein canfurther comprise one or more pharmaceutical excipients, for exampleascorbic acid, EDTA dihydrate, glycerin, citric acid monohydrate, sodiumcitrate dihydrate, sodium benzoate, sodium propionate, 70% sorbitolsolution, sucralose, FD&C Yellow #6, artificial orange flavor,artificial peppermint flavor, purified water, or any combinationthereof. In some instances, the one or more pharmaceutical excipientscomprise ascorbic acid, EDTA dihydrate, glycerin, citric acidmonohydrate, sodium citrate dihydrate, proplyparaben, methylparaben,propylene glycol, 70% sorbitol solution, sucralose, FD&C Yellow #6,artificial orange flavor, artificial peppermint flavor, purified water,or any combination thereof.

Suitable preservatives non-restrictively include mercury-containingsubstances such as phenylmercuric salts (e.g., phenylmercuric acetate,borate and nitrate) and thimerosal; stabilized chlorine dioxide;quaternary ammonium compounds such as benzalkonium chloride,cetyltrimethylammonium bromide and cetylpyridinium chloride;imidazolidinyl urea; parabens such as methylparaben, ethylparaben,propylparaben and butylparaben, and salts thereof; phenoxyethanol;chlorophenoxyethanol; phenoxypropanol; chlorobutanol; chlorocresol;phenylethyl alcohol; disodium EDTA; and sorbic acid and salts thereof.

One or more acceptable pH adjusting agents and/or buffering agents canbe included in a composition disclosed herein, including acids such asacetic, boric, citric, lactic, phosphoric and hydrochloric acids; basessuch as sodium hydroxide, sodium phosphate, sodium borate, sodiumcitrate, sodium acetate, sodium lactate andtris-hydroxymethylaminomethane; and buffers such as citrate/dextrose,sodium bicarbonate and ammonium chloride. Such acids, bases and buffersare included in an amount required to maintain pH of the composition ina pharmaceutically acceptable range.

In some instances, an antibiotic composition disclosed herein cancomprise a pH adjusting agent. In some instances, the pH adjusting agentcan be selected from the group consisting of ascorbic acid, sodiumascorbate, tartaric acid, sodium tartrate, potassium tartrate, calciumtartrate, lithium tartrate, citric acid, sodium citrate, potassiumcitrate, calcium citrate, lithium citrate, phosphoric acid, sodiumdihydrogenphosphate, sodium monohydrogenphosphate, lithium phosphate,potassium phosphate, calcium phosphate, sodium carbonate, sodiumhydrogencarbonate, lactic acid, sodium lactate, potassium lactate,calcium lactate, acetic acid, sodium acetate, potassium acetate, calciumacetate, propionic acid, sulphuric acid, sodium sulphate, potassiumsulphate, boric acid, sodium borate, maleic acid, lithium maleate,sodium maleate, potassium maleate, calcium maleate, succinic acid,lithium succinate, sodium succinate, potassium succinate, calciumsuccinate, fumaric acid, glutamic acid, formic acid, malic acid,hydrochloric acid, nitric acid, sodium hydroxide, potassium hydroxide,triethanolamine, diisopropanolamine, ammonia solution, monoethanoleamine, diethanoleamine, triethanoleamine meglumine, sodium citrate,sodium bicarbonate, potassium bicarbonate, and any combination thereof.In some instances, a pH adjusting agent disclosed herein can be aceticacid; adipic acid; ammonium aluminum sulphate; ammonium bicarbonate;ammonium carbonate; ammonium citrate, dibasic; ammonium citrate,monobasic; ammonium hydroxide; ammonium phosphate, dibasic; ammoniumphosphate, monobasic; calcium acetate; calcium acid pyrophosphate;calcium carbonate; calcium chloride; calcium citrate; calcium fumarate;calcium gluconate; calcium hydroxide; calcium lactate; calcium oxide;calcium phosphate, dibasic; calcium phosphate, monobasic; calciumphosphate, tribasic; calcium sulphate; carbon dioxide; citric acid;cream of tartar; fumaric acid; gluconic acid; glucono-delta-lactone;hydrochloric acid; lactic acid; magnesium carbonate; magnesium citrate;magnesium fumarate; magnesium hydroxide; magnesium oxide; magnesiumphosphate; magnesium sulphate; malic acid; manganese sulphate;metatartaric acid; phosphoric acid; potassium acid tartrate; potassiumaluminum sulphate; potassium bicarbonate; potassium carbonate; potassiumchloride; potassium citrate; potassium fumarate; potassium hydroxide;potassium lactate; potassium phosphate, dibasic; potassium phosphate,tribasic; potassium sulphate; potassium tartrate; potassiumtripolyphosphate; sodium acetate; sodium acid pyrophosphate; sodium acidtartrate; sodium aluminum phosphate; sodium aluminum sulphate; sodiumbicarbonate; sodium bisulphate; sodium carbonate; sodium citrate; sodiumfumarate; sodium gluconate; sodium hexametaphosphate; sodium hydroxide;sodium lactate; sodium phosphate, dibasic; sodium phosphate, monobasic;sodium phosphate, tribasic; sodium potassium hexametaphosphate; sodiumpotassium tartrate; sodium potassium tripolyphosphate; sodiumpyrophosphate, tetrabasic; sodium tripolyphosphate; sulphuric acid;sulphurous acid; tartaric acid; or any combination thereof.

In some instances, an antibiotic composition disclosed herein cancomprise a sugar alcohol. In some instances, the sugar alcohol can beselected from the group consisting of mannitol, glycerol, galactitol,fucitol, inositol, volemitol, maltotriitol, maltoetetraitol,polyglycitol, erythritol, threitol, ribitol, arabitol, xylitol, allitol,dulcitol, glucitol, sorbitol, altritol, iditol, maltitol, lactitol,isomalt, and any combination thereof. In some instances, the sugaralcohol has 3, 4, 5, 6, 7, 12, 18, or 24 carbons.

In some instances, a composition disclosed herein can comprise suitableadditives, including, but not limited to, diluents, binders,surfactants, lubricants, glidants, coating materials, plasticizers,coloring agents, flavoring agents, or pharmaceutically inert materials.Examples of diluents include, for example, cellulose; cellulosederivatives such as microcrystalline cellulose and the like; starch;starch derivatives such as corn starch, cyclodextrin and the like;sugar; sugar alcohol such as lactose, D-mannitol and the like; inorganicdiluents such as dried aluminum hydroxide gel, precipitated calciumcarbonate, magnesium aluminometasilicate, dibasic calcium phosphate andthe like. Examples of binders include, for example,hydroxypropylcellulose, methylcellulose, hydroxypropylmethylcellulose(hydroxypropyl methylcellulose), povidone, dextrin, pullulane,hydroxypropyl starch, polyvinyl alcohol, scacia, agar, gelatin,tragacanth, macrogol and the like. Examples of surfactants include, forexample, sucrose esters of fatty acids, polyoxyl stearate,polyoxyethylene hydrogenated castor oil, polyoxyethylenepolyoxypropylene glycol, sorbitan sesquioleate, sorbitan trioleate,sorbitan monostearate, sorbitan monopalmitate, sorbitan monolaurate,polysorbate, glyceryl monostearate, sodium lauryl sulfate,lauromacrogol, quaternary ammonium salts (e.g.,Benzyldimethyltetradecylammonium Chloride Hydrate, BenzethoniumChloride, Benzylcetyldimethylammonium Chloride Hydrate,Benzyldimethylstearylammonium Chloride Hydrate,Benzyldodecyldimethylammonium Chloride Dihydrate,Benzyldodecyldimethylammonium Bromide), and the like. Examples oflubricants include, for example, stearic acid, calcium stearate,magnesium stearate, talc and the like. Examples of glidants include, forexample, dried aluminum hydroxide gel, magnesium silicate and the like.Examples of coating materials include, for example, hydroxypropylmethylcellulose 2910, aminoalkyl methacrylate copolymer E, polyvinylacetaldiethylaminoacetate, macrogol 6000, titanium oxide and the like.Examples of plasticizers include, for example, triethyl citrate,triacetin, macrogol 6000 and the like.

Dosage Forms

In some instances, active agents disclosed herein are formulated as adosage form of tablet, capsule, gel, lollipop, parenteral, intraspinalinfusion, inhalation, spray, aerosol, transdermal patch, iontophoresistransport, absorbing gel, liquid, liquid tannate, suppositories,injection, I.V. drip, or a combination thereof to treat subjects. Insome instances, the agents are formulated as single oral dosage formsuch as a tablet, capsule, cachet, soft gelatin capsule, hard gelatincapsule, extended release capsule, tannate tablet, oral disintegratingtablet, multi-layer tablet, effervescent tablet, bead, liquid, oralsuspension, chewable lozenge, oral solution, lozenge, lollipop, oralsyrup, sterile packaged powder including pharmaceutically-acceptableexcipients, other oral dosage forms, or a combination thereof. In someinstances, a composition of the disclosure herein can be administeredusing one or more different dosage forms which are further disclosedherein. For example, a composition comprising multiple active agents canbe administered in solid, semi-solid, micro-emulsion, gel, patch orliquid form. Such dosage forms are further disclosed herein. In someinstances, the disclosure herein relates to methods and compositionsformulated for oral delivery to a subject in need. In some instances, acomposition can be formulated so as to deliver one or morepharmaceutically active agents to a subject through a mucosa layer inthe mouth or esophagus. In some instances, the composition can beformulated to deliver one or more pharmaceutically active agents to asubject through a mucosa layer in the stomach and/or intestines.

In some instances, compositions disclosed herein are provided inmodified release dosage forms (such as immediate release, controlledrelease or both), which comprise an effective amount of an active agent;and one or more release controlling excipients as disclosed herein.Suitable modified release dosage vehicles include, but are not limitedto, hydrophilic or hydrophobic matrix devices, water-soluble separatinglayer coatings, enteric coatings, osmotic devices, multi-particulatedevices, and combinations thereof. In some instances, the compositionscomprise non-release controlling excipients. In some instances,compositions disclosed herein are provided in enteric coated dosageforms. In some instances, compositions disclosed herein comprisenon-release controlling excipients. In some instances, compositionsdisclosed herein are provided in effervescent dosage forms. In someinstances, the compositions comprise non-release controlling excipients.

In some instances, a composition disclosed herein can be provided in adosage form that has at least one component that facilitates theimmediate release of an active agent, and at least one component thatcan facilitate the controlled release of an active agent. In someinstances, the dosage form can be capable of giving a discontinuousrelease of the compound in the form of at least two consecutive pulsesseparated in time from 0.1 up to 24 hours. The compositions can compriseone or more release controlling and non-release controlling excipients,such as those excipients suitable for a disruptable semi-permeablemembrane and as swellable substances. In some instances, a compositiondisclosed herein can be provided in a dosage form for oraladministration to a subject, which comprise one or more pharmaceuticallyacceptable excipients or carriers, enclosed in an intermediate reactivelayer comprising a gastric juice-resistant polymeric layered materialpartially neutralized with alkali and having cation exchange capacityand a gastric juice-resistant outer layer. In some instances, thecompositions further comprise cellulose, disodium hydrogen phosphate,hydroxypropyl cellulose, hypromellose, lactose, mannitol, or sodiumlauryl sulfate. In some instances, the compositions further compriseglyceryl monostearate 40-50, hydroxypropyl cellulose, hypromellose,magnesium stearate, methacrylic acid copolymer type C, polysorbate 80,sugar spheres, talc, or triethyl citrate. In some instances, thecompositions further comprise carnauba wax, crospovidone, diacetylatedmonoglycerides, ethylcellulose, hydroxypropyl cellulose, hypromellosephthalate, magnesium stearate, mannitol, sodium hydroxide, sodiumstearyl fumarate, talc, titanium dioxide, or yellow ferric oxide. Insome instances, the compositions further comprise calcium stearate,crospovidone, hydroxypropyl methylcellulose, iron oxide, mannitol,methacrylic acid copolymer, polysorbate 80, povidone, propylene glycol,sodium carbonate, sodium lauryl sulfate, titanium dioxide, and triethylcitrate.

In some instances, compositions disclosed herein are in unit-dosageforms or multiple-dosage forms. Unit-dosage forms, as used herein, referto physically discrete units suitable for administration to human ornon-human animal subjects and packaged individually. Each unit-dosecontains a predetermined quantity of an active ingredient(s) sufficientto produce the desired therapeutic effect, in association with therequired pharmaceutical carriers or excipients. Examples of unit-dosageforms include, but are not limited to, ampules, syringes, andindividually packaged tablets and capsules. In some instances,unit-dosage forms are administered in fractions or multiples thereof. Amultiple-dosage form can be a plurality of identical unit-dosage formspackaged in a single container, which can be administered in segregatedunit-dosage form. Examples of multiple-dosage forms include, but are notlimited to, vials, bottles of tablets or capsules, or bottles of pintsor gallons. In some instances, the multiple dosage forms comprisedifferent pharmaceutically active agents.

In some instances, a kit can be provided comprising an antibioticcomposition disclosed herein. In some instances, the kit further cancomprise a set of instructions.

In some instances, compositions disclosed herein can be formulated indosage forms for oral, parenteral, or topical administration. In someinstances, the compositions can be formulated as a modified releasedosage form, including immediate-, delayed-, extended-, prolonged-,sustained-, pulsatile-, controlled-, extended, accelerated- and fast-,targeted-, programmed-release, and gastric retention dosage forms. Insome instances, the compositions can be in one or more dosage form. Forexample, a composition can be administered in a solid or liquid form.Examples of solid dosage forms include but are not limited to discreteunits in capsules or tablets, as a powder or granule, or present in atablet conventionally formed by compression molding. In some instances,such compressed tablets are prepared by compressing in a suitablemachine the three or more agents and a pharmaceutically acceptablecarrier. The molded tablets can be optionally coated or scored, havingindicia inscribed thereon and can be so formulated as to causeimmediate, substantially immediate, slow, controlled or extended releaseof the active agents disclosed herein. In some instances, dosage formsdisclosed herein comprise acceptable carriers or salts known in the art,such as those described in the Handbook of Pharmaceutical Excipients,American Pharmaceutical Association (1986), incorporated by referenceherein in its entirety. In some instances, one or more pharmaceuticallyactive agents are mixed with a pharmaceutical excipient to form a solidpreformulation composition comprising a homogeneous mixture of compoundsdisclosed herein. When referring to compositions disclosed herein as“homogeneous”, it can be meant that the agents are dispersed evenlythroughout the composition so that the composition can be subdividedinto unit dosage forms such as tablets or capsules. In some instances,this solid preformulation composition can be then subdivided into unitdosage forms of the type described above comprising from, for example,about 1.0 mg to about 15 mg of an active agent disclosed herein.

In some instances, compositions disclosed herein are formulated, in thecase of capsules or tablets, to be swallowed whole, for example withwater. The inclusion of the side-effect-reducing agent such as anantihistamine or antiemetic to abate common symptoms of nausea andvomiting are believed beneficial in that promethazine or a salt thereof,or the like will eliminate or minimize the amount of discomfort. Adverseeffects reduced or eliminated include but are not limited to nausea,vomiting, other gastric upsets, constipation, skin rashes, allergicreactions such as swelling, difficulty breathing, closing of throat,abdominal pain, unusual bleeding or bruising, CNS suppression andrespiratory suppression.

In some instances, a dosage form disclosed herein can be manufacturedusing processes that are well known to those of skill in the art. Forexample, for the manufacture of tablets (including but not limited tosingle layer, bi-layer, coated, of multi-layer tablets) or capsules, theagents can be dispersed uniformly in one or more excipients, forexample, using high shear granulation, low shear granulation, fluid bedgranulation, or by blending for direct compression. Excipients includediluents, binders, disintegrants, dispersants, lubricants, glidants,stabilizers, surfactants and colorants. Diluents, also termed “fillers”,are used to increase the bulk of a tablet so that a practical size canbe provided for compression. Non-limiting examples of diluents includelactose, cellulose, microcrystalline cellulose, mannitol, dry starch,hydrolyzed starches, powdered sugar, talc, sodium chloride, silicondioxide, titanium oxide, dicalcium phosphate dihydrate, calcium sulfate,calcium carbonate, alumina and kaolin. In some instances, binders impartcohesive qualities to a tablet formulation, or a particle formulation ina capsule, and are used to help a tablet remain intact aftercompression. Non-limiting examples of suitable binders include starch(including corn starch and pregelatinized starch), gelatin, sugars(e.g., glucose, dextrose, sucrose, lactose and sorbitol), celluloses,polyethylene glycol, waxes, natural and synthetic gums, e.g., acacia,tragacanth, sodium alginate, and synthetic polymers such aspolymethacrylates and polyvinylpyrrolidone. In some instances,lubricants facilitate tablet manufacture; non-limiting examples thereofinclude magnesium stearate, calcium stearate, stearic acid, glycerylbehenate, and polyethylene glycol. In some instances, disintegrantsfacilitate tablet disintegration after administration, and non-limitingexamples thereof include starches, alginic acid, crosslinked polymerssuch as, e.g., crosslinked polyvinylpyrrolidone, croscarmellose sodium,potassium or sodium starch glycolate, clays, celluloses, starches, gumsand the like. Non-limiting examples of suitable glidants include silicondioxide, talc and the like. In some instances, stabilizers inhibit orretard drug decomposition reactions, including oxidative reactions. Insome instances, a surfactant can be anionic, cationic, amphoteric ornonionic. In some instances, the tablets (or particles) comprisenontoxic auxiliary substances such as pH buffering agents,preservatives, e.g., antioxidants, wetting or emulsifying agents,solubilizing agents, coating agents, flavoring agents, and the like. Insome instances, exemplary excipients include cellulose ethers such ashydroxypropylmethylcellulose (e.g., Methocel K4M) or silicifiedmicrocrystalline cellulose; polyvinylacetate-based excipients such as,e.g., Kollidon SR, and polymers and copolymers based on methacrylatesand methacrylic acid such as, e.g., Eudragit NE 30D; microcrystallinecellulose, sodium carboxymethyl cellulose, sodium starch glycolate, cornstarch, colloidal silica, Sodium Laurel Sulphate, Magnesium Stearate,Prosolve SMCC (HD90), croscarmellose Sodium, Crospovidone NF, AvicelPH200 or a combination thereof. In some instances, compositionsdisclosed herein comprise one or more combination of excipients thatslow the release of the agents by coating or temporarily bonding ordecreasing their solubility of the active agents. Examples of theseexcipients include cellulose ethers such as hydroxypropylmethylcellulose(e.g., Methocel K4M) or silicified microcrystalline cellulose,polyvinylacetate-based excipients such as, e.g., Kollidon SR, andpolymers and copolymers based on methacrylates and methacrylic acid suchas, e.g., Eudragit NE 30D.

In some instances, compositions comprise one or more carriers thatprotect the agents against rapid elimination from the body, such astime-release formulations or coatings. Such carriers includecontrolled-release formulations, including, for example,microencapsulated delivery systems. In some instances, the active agentsare included in the pharmaceutically acceptable carrier in amountssufficient to treat a subject's pain, with reduced adverse effects. Insome instances, the compositions are in oral-dosage form and comprise amatrix that includes, for example, an active agent formulated forcontrolled release. In some instances, the matrix can be compressibleinto a tablet and can be optionally overcoated with a coating thatcontrols the release of the active agent from the composition. In someinstances, blood levels of analgesics are maintained within atherapeutic range over an extended period of time. In certain someinstances, the matrix can be encapsulated. Tablets or capsulescontaining a composition disclosed herein can be coated or otherwisecompounded to provide a dosage form affording the advantage of prolongedaction. For example, the tablet or capsule contains an inner dosage andan outer dosage component, the latter being in the form of an envelopeover the former. The two components can be separated by an enteric layerthat serves to resist disintegration in the stomach and permit the innercomponent to pass intact into the duodenum or to be controlled inrelease. In some instances, for controlled extended release, the capsulehas micro drilled holes. In some instances, a coating comprising aside-effect-reducing compound can be prepared by admixing a compoundlike promethazine with polyvinylpyrrolidone (PVP) 29/32 or hydroxypropylmethylcellulose (HPMC) and water/isopropyl alcohol and triethyl acetate.Such a coating can be spray coated onto the tablet cores. In someinstances, the coating can be applied using a press-coating process witha blend consisting of 80% by weight promethazine and 20% by weight oflactose and hydroxypropyl methylcellulose type 2910. Press-coatingtechniques are known in the art and are described in U.S. Pat. No.6,372,254, which can be herein incorporated by reference in itsentirety.

In some instances, a dosage form disclosed herein can be an effervescentdosage form. Effervescent means that the dosage form, when mixed withliquid, including water and saliva, evolves a gas. Some effervescentagents (or effervescent couple) evolve gas by means of a chemicalreaction which takes place upon exposure of the effervescentdisintegration agent to water and/or to saliva in the mouth. Thisreaction can be the result of the reaction of a soluble acid source andan alkali monocarbonate or carbonate source. The reaction of these twogeneral compounds produces carbon dioxide gas upon contact with water orsaliva. An effervescent couple (or the individual acid and baseseparately) can be coated with a solvent protective or enteric coatingto prevent premature reaction. In some instances, such a couple can bemixed with previously lyophilized particles (such as one or morepharmaceutically active agents coated with a solvent protective orenteric coating. In some instances, the acid source can be any which aresafe for human consumption and includes food acids, acid and hydriteantacids such as, for example: citric, tartaric, amalic, fumeric,adipic, and succinics. Carbonate sources include dry solid carbonate andbicarbonate salt such as, for example, sodium bicarbonate, sodiumcarbonate, potassium bicarbonate and potassium carbonate, magnesiumcarbonate and the like. Reactants which evolve oxygen or other gassesand which are safe for human consumption are also included. In someinstances, citric acid and sodium bicarbonate can be used.

In some instances, a dosage form disclosed herein can be in a candy form(e.g., matrix), such as a lollipop or lozenge. In some instances, one ormore pharmaceutically active agents are dispersed within a candy matrix.In some instances, the candy matrix can comprise one or more sugars(such as dextrose or sucrose). In some instances, the candy matrix canbe a sugar-free matrix. The choice of a particular candy matrix can besubject to wide variation. In some instances, conventional sweetenerssuch as sucrose are utilized, or sugar alcohols suitable for use withdiabetic patients, such as sorbitol or mannitol might be employed. Insome instances, other sweeteners, such as the aspartanes, are easilyincorporated into a composition in accordance with compositionsdisclosed herein. The candy base can be very soft and fast dissolving,or can be hard and slower dissolving. Various forms will have advantagesin different situations. In some instances, a candy mass comprising atleast one pharmaceutically active agent can be orally administered to asubject in need thereof so that the agent will be released into thesubject's mouth as the candy mass dissolves. The drug rapidly enters thesubject bloodstream, and importantly, the blood in the veins drainingfrom the mouth and the pharyngeal and esophageal areas passes through asubstantial portion of the body (so that the drug can be absorbed)before the blood passes through the liver (where the drug can beinactivated). In some instances, a subject in need thereof can be ahuman adult or child in suffering from a cough and/or pain. In someinstances, a candy matrix (e.g., lollipop or lozenge) disclosed hereincan comprise a composition that lacks a stimulant. In some instances,the composition has a sedative effect in addition to providing coughand/or pain relief to a subject in need thereof. In some instances, thecandy matrix (lollipop or lozenge) can comprise a composition that cancomprise a stimulant. In some instances, the composition provides ananti-sedative effect in addition to providing cough and/or pain reliefto a subject in need thereof. In some instances, a candy mass disclosedherein can comprise one or more layers which comprise differentpharmaceutically active agents and or rates of dissolution. In someinstances, a multilayer candy mass (such as a lollipop) can comprise anouter layer with a concentration of one or more pharmaceutically activeagents differing from that of one or more inner layers. Such a drugdelivery system has a variety of applications. By way of example, it canbe desirable to quickly get a predetermined dose of a firstpharmaceutically active agent into the bloodstream to obtain a desiredeffect and then use a different inner layer to deliver one or more otheragents. The choices of matrix and the concentration of the drug in thematrix are important factors with respect to the rate of drug uptake. Insome instances, a matrix that dissolves quickly delivers drug into thepatient's mouth for absorption more quickly than a matrix that can beslow to dissolve. In some instances, a candy matrix that contains one ormore pharmaceutically active agents in a high concentration releasesmore of the one or more pharmaceutically active agents in a given periodof time than a candy having a low concentration.

In some instances, dosage forms disclosed herein take the form ofpharmaceutical particles manufactured by a variety of methods, includingbut not limited to high-pressure homogenization, wet or dry ballmilling, or small particle precipitation (e.g., nGimat's NanoSpray).Other methods useful to make a suitable powder formulation are thepreparation of a solution of active ingredients and excipients, followedby precipitation, filtration, and pulverization, or followed by removalof the solvent by freeze-drying, followed by pulverization of the powderto the desired particle size. In some instances, the pharmaceuticalparticles have a final size of 3-1000 μm, such as at most 3, 4, 5, 6, 7,8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350,400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000 μm. Insome instances, the pharmaceutical particles have a final size of 10-500μm. In some instances, the pharmaceutical particles have a final size of50-600 μm. In some instances, the pharmaceutical particles have a finalsize of 100-800 μm. In some instances, these dosage forms includeimmediate-release particles in combination with controlled-releaseparticles in a ratio sufficient useful for delivering the desireddosages of active agents.

Liquid Compositions

In some aspects, liquid compositions disclosed herein are shelf-stable,for examples, the liquid compositions do not separate on the shelf (bothfloating and settling) or do not require vigorous shaking (which greatlyaffects dosing consistency). In some instances, one or more activeagents in liquid compositions disclosed herein are provided as inmodified release, e.g., controlled release, immediate release, or mixed.In some instances, one or more active agents in liquid compositionsdisclosed herein can comprise a decongestant, antitussive, expectorantor analgesic in a matrix formulated for modified release. Exemplaryexpectorants include ammonium chloride, N-acetylcysteine, ambroxol,guaifenesin (e.g., glycerol, guaiacolate), terpin hydrate, glycerylguaiacolate, potassium iodide, potassium citrate, potassiumguaicolsulfonate, Oregano Leaf Extract 25-500 mg (which can be a liquidextract), Red Clover 25-500 mg, Buckthorn Root 25-500 mg, Fenugreek25-500 mg, or any mixture thereof. Examples of carriers for the activesinclude any degradable, partially degradable or non-degradable andgenerally biocompatible polymer, e.g., polystirex, polypropylene,polyethylene, polacrilex, poly-lactic acid (PLA), polyglycolic acid(PGA) and/or poly-lactic polyglycolic acid (PGLA), e.g., in the form ora liquid, matrix, or bead.

In some instances, a liquid composition disclosed herein has a viscosity(spindle viscosity) from about 150 to about 1000 centipoises at 50 RPM,for example from about 200 to about 1000 centipoises at 50 RPM or fromabout 400 to about 700 centipoises at 50 RPM; or from about 150 to about1200 centipoises measured at 10 RPM. Viscosity can be measured by amethod in USP (United States Pharmacopeia), selected from <911>Viscosity-Capillary Viscometer Methods, <912> Rotational RheometerMethods, and/or <913> Rolling Ball Viscometer Method. In some instances,an amount of viscosity modifier used depends on the desired “thickness”of the composition and the type viscosity modifier used. In someinstances, combinations of viscosity modifiers are employed. Forexample, in an exemplary embodiment with a viscosity of about 1500 toabout 4500 cps, up to about 1.0 w/v xanthan gum can be used with up toabout 3.0 w/v microcrystalline cellulose can be as a viscosity modifier.In some instances, a pH of a liquid composition disclosed herein can beabout: 2.5-5, 6-8, 5-9, 4-10, 7-8, 7-9, 7-10, 6-7, 5-7, or 4-7. In someinstances, a pH of a liquid composition disclosed herein can be about:2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12. In some instances, the pH can beabout 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2,7.3, 7.4, 7.5, 7.8, 7.9, 7.10, for example, ranging from about 6.8 toabout 7.4.

In one aspect, a liquid composition disclosed herein can be a suspensioncomprising beads (e.g., microbeads), wherein a portion of the one ormore beads have an immediate release profile and another portion have acontrolled release profile. In some instances, one or more beads includean enteric coat, a resin coat, a lacquer coat, a pH-sensitive coating, abiodegradable polymer matrix, a water soluble matrix, an ionic matrix,or any combination thereof. In some instances, one or more beads includeone or more polymers selected from cellulose, ethylcellulose,methylcellulose, propylcellulose, methoxypropylcellulose, cellulosenitrate, poly(vinyl alcohol), poly(vinyl chloride), polystyrene,polyethylene, polypropylene, poly(ethylene-co-vinyl acetate),poly(hydroxybutyric acid), poly(hydroxyvalerianic acid-co-hydroxybutyricacid), poly(lactic acid), poly(glycolic acid), poly(lacticacid-co-glycolic acid), poly(.epsilon.(-caprolactones),poly(.epsilon.-caprolactone-co-DL-lactic acid), poly(maleic anhydride),polyamides, gelatin, chitosan, collagen,poly(hydroxyalkyl)-L-glutamines,poly(.gamma.-ethyl-L-glutaminate-co-glutamic acid),poly(L-leucine-co-L-aspartic acid), poly(proline-co-glutamic acid),poly(alkyl 2-cyanoacrylates), polyurethanes, poly(methyl methacrylate),poly(methyl methacrylate-co-methacrylic acid) andpoly(methacrylate-co-hydroxypropyl methacrylate), polystyrene,polistirex, polacrilex, salts thereof, and any combination thereof.

In some instances, a liquid composition disclosed herein can be a syrup,a ready-to-use suspension, or extemporaneously prepared liquid syrup orsuspension such as, for example, dry powder for reconstitution withwater, liquid concentrate for dilution, dispersible tablet or capsule.In the case of extemporaneously prepared syrup or suspension, theconcentration of ingredients can be based on the reconstituted product.In some instances, the liquid dosing form can be for oraladministration, intravenous injection, intramuscular injection, or fortopical administration (e.g., as a cream, gel, ointment, or bandage). Anorally administered liquid dosing form can be beneficial for subjectsthat have dysphagia or difficulty swallowing. In some instances, theliquid dosage form includes one or more pharmaceutically acceptablecarriers or excipients. In some instances, the liquid pharmaceuticalcomposition contains one or more active agents, e.g., present attherapeutically effective amounts in the dosage form. These amountsdiffer depending on the drug and prescribed dosage regimens. Forinstance, liquid preparations intended for infants contain high drugconcentrations to enable small doses and reduced dosing frequency. Insome instances, an amount of drug in the composition can be from about0.02 to about 15 percent by weight, e.g., from about 0.1 to about 10percent by weight of the total composition. In the case of dry powderfor reconstitution with water, the drug can be present as uncoated orcoated particles.

In some instances, a liquid composition disclosed herein can comprise ataste masking liquid excipient base for administration of an unpleasanttasting medicine. In some instances, said excipient base has higher thannormal viscosities, e.g., due to presence of polyethylene glycol and/orsodium carboxymethyl cellulose. In some instances, high viscosity liquidexcipient base provides taste masking benefits to the extent that extrastrength compositions can be prepared containing increasedconcentrations of adverse tasting pharmaceutical compositions. Forexample, an active agent normally administered in dosages of no morethan 100 milligrams in 5 milliliters of liquid, can be administered indosages of 200 milligrams in the same volume of liquid without thepatient experiencing an unduly adverse taste.

In another aspect, a method disclosed herein increases the shelf-lifeand stability of the actives agents, e.g., by preventing the separationof the components by taking steps to reduce or eliminate bubbleformation. In some instances, steps for minimizing, reducing and/oreliminating bubble formation include, but are not limited to using thefollowing steps alone or in combination: using a diaphragm pump tocombine, e.g., the water and the thixotropic agent and one or morepreservatives, colorants and flavorants; placing the recirculating tubebelow the surface of the liquid; adding liquids along the side of avessel holding the liquid; sprinkling beads (e.g., one or more beadsthat includes one or more active agents) onto the surface of the liquid;mixing the solution in the absence of one or more paddles that scrapethe vessel; mixing the solution with a propeller mixer; mixing thesolution with a propeller mixer at a speed that reduces or minimizescavitation and combinations of two or more of these steps.

In some instances, a liquid composition disclosed herein can be for usein treating a disease or condition disclosed herein, e.g., cough,allergy, cold, or associated symptoms. In some instances, a subject(e.g., person) suffering from cold or cold-like symptoms finds relief byorally ingesting a safe and effective amount of the liquid compositionsas described above. The safe and effective amount of liquid compositioncan be dependent upon the concentration of the therapeutic componentspresent in the liquid compositions. In some instances, the safe andeffective amount of liquid composition can be in a range of 1-30 ml,e.g. 1-10 ml, per dosage of the liquid composition. In some instances, aliquid composition disclosed herein can be safely consumed by a child.In some instances, a subject (e.g., person) can be taking multiple doses(1, 2, 3, 4, 5, 6, 7, or 8 doses) of the liquid composition per day. Insome instances, a liquid composition herein provides an effective amountof one or more active agents for 2-4 hours, 4-6 hours, 6-8 hours, 12hours, or 24 hours. In some instances, a liquid composition disclosedherein can be administered to a subject (e.g., person) in a liquid form(e.g. by dosage cup or reservoir such as a spoon) or it can beencapsulated in a soft gelatin capsule that can be chewable orswallowable by the individual. In some instances, the liquid compositioncan be blended with compositions such as ice, milk, soda, juice, or someother edible composition and administered to the individual.

In some instances, a liquid dosage form disclosed herein can be for oraladministration, intravenous injection, intramuscular injection, or fortopical administration (e.g., as a cream or gel). An orally administeredliquid dosing form can be beneficial for subjects that have dysphagia ordifficulty swallowing. A single dose of an orally administered liquiddosing form can be from 1 mL to about 500 mL in volume, or more. Forexample, the single dose of an orally administered liquid dosing formcan be about 1-500 mL, 1-250 mL, 1-100 mL, 1-50 mL, 1-30 mL, 1-20 mL,1-15 mL, 1-10 mL, 1-5 mL, 1-2.5 mL, 2.5-50 mL, 2.5-30 mL, 2.5-20 mL,2.5-15 mL, 2.5-10 mL, 2.5-5 mL, 5-50 mL, 5-30 mL, 5-20 mL, 5-15 mL, 5-10mL, 10-50 mL, 10-30 mL, 10-20 mL, 10-15 mL, 15-50 mL, 15-30 mL, 15-20mL, 20-50 mL, 20-30 mL, 30-50 mL, 1 mL, 1.5 mL, 2 mL, 2.5 mL, 3 mL, 3.5mL, 4 mL, 4.5 mL, 5 mL, 5.5 mL, 6 mL, 6.5 mL, 7 mL, 7.5 mL, 8 mL, 8.5mL, 9 mL, 9.5 mL, 10 mL, 11 mL, 12 mL, 13 mL, 14 mL, 15 mL, 16 mL, 17mL, 18 mL, 19 mL, 20 mL, 21 mL, 22 mL, 23 mL, 24 mL, 25 mL, 30 mL, 35mL, 40 mL, 45 mL, 50 mL, 60 mL, 70 mL, 80 mL, 90 mL, 100 mL, 110 mL, 120mL, 130 mL, 140 mL, 150 mL, 160 mL, 170 mL, 180 mL, 190 mL, 200 mL, 250mL, 300 mL, 350 mL, 400 mL, 450 mL, or 500 mL.

In some instances, pharmaceutically acceptable carriers or excipientsdisclosed herein include pH modifying agent(s) (e.g., bufferingagent(s)), stabilizing agent(s), thickening agent(s), sweeteningagent(s), flavoring agent(s), colorant agent(s), preservative agent(s),emulsifying agent(s), solubilizing agent(s), antioxidant agent(s), orany combination thereof.

In some instances, a stabilizing agent disclosed herein includes anysuitable monohydroxy phenol component or polyhydroxy phenol component,or a combination thereof. In some instances, such stabilizing agents arealso function as antioxidant agents, or antimicrobial agents. In someinstances, a thickening agent or viscosity-enhancing agent disclosedherein improves the mouth-feel of the liquid oral dosage form and/or tohelp coat the lining of the gastrointestinal tract. Exemplary thickeningagents include acacia, alginic acid bentonite, carbomer,carboxymethylcellulose calcium or sodium, cetostearyl alcohol, methylcellulose, ethylcellulose, glycerin, gelatin guar gum, hydroxyethylcellulose, hydroxymethyl cellulose, hydroxypropyl cellulose,hydroxypropyl methyl cellulose (“HPMC”), any other suitablecellulose-based component, maltodextrin, polyvinyl alcohol, povidone,propylene carbonate, propylene glycol alginate, sodium alginate, sodiumstarch glycolate, starch tragacanth, and xanthan gum, or a combinationthereof. Thickening agent, when included, can be present in an amount ofabout 0.1 volume percent to 20 volume percent (v/v), based on the totalvolume of the solution. In one example, glycerin can be present in anamount of about 1 volume percent to 10 volume percent (v/v), based onthe total volume of the solution. Exemplary amounts of thickening agentinclude from about 1 volume percent to 12 volume percent (v/v), andpreferably at an amount of about 4 volume percent to 10 volume percent(v/v), based on the total volume of the solution. An exemplary amountincludes about 6 to 10 volume percent (v/v). In some instances, asweetening agent can be optionally included in the oral liquid dosageform. Exemplary sweetening agents include sorbitol, saccharin,acesulfame, e.g., acesulfame potassium, sucralose, xylitol, maltitol,sucrose, aspartame, fructose, neotame, glycerin, sodium saccharate,glycyrrhizin dipotassium, acesulfame K, mannitol, invert sugar, andcombinations thereof, or components containing a sweetening agent, suchas one or more sucralose-containing components or saccharin-containingcomponents, can be added to modify the taste of the composition.Alternatively, or in addition, a viscous sweetener such as one or moreof a sorbitol solution, a syrup (sucrose solution), or high-fructosecorn syrup can be used and, in addition to sweetening effects, can beuseful to increase viscosity and to retard sedimentation. In someinstances, the sweetening agent includes an acesulfame-containing,sucralose-containing, or saccharin-containing component. In someinstances, the sweetening agent includes glycerin, saccharin, liquidsugar (sucrose solution), or a combination thereof. Such a sweeteningagent, if present, can be present in an amount sufficient to minimize ormask any off-flavors in the taste of the active agents, and also tominimize or mask any other off-flavor components included in theformulation if desired. Sweetening agent(s), when included, can bepresent in an amount of about 0.1 volume percent to 85 volume percent(v/v), based on the total volume of the solution. In one example, thesweetening agent can be present in an amount of about 5 volume percentto 70 volume percent (v/v), based on the total volume of the solution.Exemplary amounts of glyercin include about 2 volume percent to 18volume percent (v/v), preferably about 5 volume percent to 10 volumepercent (v/v). In some instances, exemplary amounts of liquid sugarinclude about 40 volume percent to 75 volume percent (v/v), preferablyabout 60 volume percent to 70 volume percent (v/v), based on the totalvolume of the solution. In some instances, certain types of thickeningagent or sweetening agent act as a solubilizing agent or a stabilizingagent, or both, or have other properties, when included as a componentof a pharmaceutically acceptable carrier. For example, a sweeteningagent such as glycerin acts as a thickening agent. In some instances, anoral liquid dosage form contains, in addition to a sweetening agent, aflavoring agent, for example, one or more of natural and artificialfruit, artificial banana, strawberry, and pineapple. In some instances,a colorant agent, when included in the liquid dosage form, can beprovided in an amount sufficient to provide the compositions with a moreaesthetic and/or distinctive appearance. Exemplary colorant agentsinclude one or more synthetic organic food additives (e.g., food dyessuch as food red dye Nos. 2 and 3, food yellow dye Nos. 4 and 5 and foodblue dye Nos. 1 and 2), water-insoluble lake dyes (e.g., aluminum saltsof the above synthetic organic food additives, etc.), and naturalpigments (e.g., beta-carotene, chlorophyll, iron oxide red, etc.). Othersuitable colorants include D&C Red No. 33, FD&C Red No. 3, FD&C Red No.40, D&C Yellow No. 10, and C Yellow No. 6, or any combination of theseor the above colorants. In some instances, suitable preservativeagent(s) are optionally included in the liquid dosage form. Thepreservative agent(s) can be in an amount sufficient to extend theshelf-life or storage stability, or both, of the liquid dosage form.Exemplary preservative agents include sodium benzoate, paraoxybenzoicacid esters, methyl, ethyl, butyl, and propyl parabens, chlorobutanol,benzyl alcohol, phenylethylalcohol, dehydroacetic acid, sorbic acid,benzalkonium chloride (BKC), benzethonium chloride, phenol,phenylmercuric nitrate, thimerosal, and a combination thereof. In someinstances, the pH of the liquid dosage form can be adjusted by abuffering agent. The buffering agent can be present in an amountsufficient to buffer the pH of the solution and minimize degradation ofthe active ingredients. In some instances, some buffering agents alsomodulate active ingredient solubility in the liquid dosage form.Exemplary buffering agents include one or more of gluconate, lactate,citrate, acetate, phosphate, benzoate, and/or carbonate salts. The pHcan be adjusted with a combination of two or more of these bufferingagents, e.g., citric acid and sodium benzoate. The buffering agent canbe present as a buffer solution. In another example, the buffering agentincludes a phosphate, such as a potassium phosphate or sodium phosphate,or a combination thereof. In some instances, emulsifying agents can beincluded in the liquid dosage form in an amount sufficient to facilitatemore uniform dispersion of one or more active ingredients or otherpharmaceutically acceptable excipient that can be not generally solublein the liquid. Exemplary emulsifying agents include gelatin, egg yolk,casein, cholesterol, acacia, tragacanth, chondrus, pectin, methylcellulose, carbomer, cetostearyl alcohol, cetyl alcohol, or acombination thereof. Solubilizing agents can be included in the liquiddosage form, e.g., in an amount sufficient to facilitate greater or morerapid dissolution of one or more active ingredients or other excipients.Exemplary solubilizing agents include an alcohol, e.g., 95% ethylalcohol, a glycol, glycerin, D-mannitol, trehalose, benzyl benzoate,trisaminomethane, cholesterol, triethanolamine, sodium carbonate, sodiumcitrate, sodium salicylate, sodium acetate, and a combination thereof.Exemplary alcohols include ethanol, isopropanol, t-butanol, phenol,cresol, a benzyl alcohol, or a combination thereof. Exemplary glycolsinclude C2-20 alkenes functionalized with a glycol, including propyleneglycol, polypropylene glycol, polyethylene glycol, etc., or acombination thereof. A solubilizing agent can be included in an amountof about 1 volume percent to 20 volume percent (v/v), or about 4 volumepercent to 15 volume percent (v/v), based on the total volume of thesolution. Exemplary amounts of solubilizing agent include about 7 volumepercent to 12 volume percent (v/v) based on the total volume of thesolution. In some instances, a stabilizing agent can be included in theliquid dosage form. Exemplary stabilizing agents include, for example,one or more liquid excipients such as ethanol, glycerin; one or moreglycols, such as polyethylene glycol, e.g., PEG-400, propylene glycol,or polypropylene glycol; a cellulose-based component, such ashydroxypropylmethylcellulose (HPMC) or hydroxymethylcellulose (HMC); orany combination thereof. In some instances, certain solubilizing agentsfunction effectively as a stabilizing agent. For example, propyleneglycol functions as both a solubilizing agent and as a stabilizingagent. In some instances, an antioxidant(s) can be included in theliquid dosage form. Exemplary antioxidants include one or moreflavonoids, anthocyanidins, anthocyanins, proanthocyanidins, andcombinations thereof. The antioxidant(s), when used, can help providelong term stability to the liquid compositions, e.g., at ambientconditions for at least about one month, preferably for at least about 3months, at least about 24 months, or longer, depending on the type andconcentration of antioxidant used and depending on other components ofthe storage microenvironment, such as pH, buffering agent, etc.

In some instances, a composition disclosed herein exhibits improved ormore desired performance with respect to drug concentration,dissolution, dispersion, stability, safety, emulsification, efficacy,flavor, patient compliance, bioavailability, and/or otherpharmacokinetic, chemical and/or physical properties. In some instances,an effective amount of one or more active agents can be dissolved togenerate a substantially stable, or stable, solution with one or more ofthe pharmaceutically acceptable carriers or excipients as disclosedherein. In some instances, an oral liquid dosage form disclosed hereincan be a controlled-release oral liquid dosage form. The controlledrelease oral liquid dosage form can provide for controlled or sustainedrelease of one or more active ingredients from a gel, matrix, capsule,or resin material, or any combination of controlled or sustained releasetechnology that can be suspended or dissolved in a liquid composition.In some instances, the controlled-release oral liquid dosage form cancomprise one or more excipients such as xanthan gum, sodium alginate,complex coacervate pairs such as gelatin or other polymers andcarrageenan, and thermal gelling methycellulose formulations. Suchexcipients can influence the dissolution and/or diffusion rate of asuspended active ingredient so as to modify the absorptioncharacteristics of the active ingredient as compared to a conventionaloral liquid dosage form. In some instances, the controlled-release oralliquid dosage form can be administered in a normally liquid compositionand only subsequently forms a semi-solid or gel-like persistent matrixin the environment of the stomach. In some instances, acontrolled-release oral liquid dosage form can comprise an aqueous,partially aqueous or non-aqueous solution or suspension of xanthan gum,sodium alginate, or sodium alginate LV (low viscosity, calciumdepleted), gelatin and carrageenan, methylcellulose, or a combinationthereof. In some instances, the controlled-release oral liquid dosageform can comprise xanthan gum (e.g., Kelco SS-4749 and othercommercially available types) in an amount of from about 0.3 to about3.0 percent by weight. In some instances, the controlled-release oralliquid dosage form can comprise xanthan gum in an amount of about 1.0percent by weight. In some instances, the controlled-release oral liquiddosage form can comprise sodium alginate in an amount of from about 0.5to about 3.0 weight percent, or from about 0.3 to about 1.5 percent byweight of each gelatin and carrageenan. In some instances, eachcarrageenan of the iota type and gelatin type B can be present at levelsof at least about 0.5 percent by weight. In some instances, thecontrolled-release oral liquid dosage form can comprise at least about 1weight percent of sodium alginate. In some instances, thecontrolled-release oral liquid dosage form can comprise methylcellulose(e.g., Type A15C, Dow Chemical Co.) in an amount of from about 1.0 toabout 3.0 weight percent. In some instances, the controlled-release oralliquid dosage form can comprise methylcellulose (e.g., Type A15C, DowChemical Co.) in an amount of about 2.0 weight percent. In someinstances, the controlled-release oral liquid dosage form comprise otherexcipients such as, for example, locust bean gum, salts such as NaCl,sugars such as sorbitol, Na3PO4, CaCO3, Ca2HPO4 and the like. Thecontrolled-release oral liquid dosage form can comprise carbonatecompounds such as calcium carbonate. The calcium carbonate can “float”the gelatinous matrix in a predetermined region of the stomach so thatit can be contacted with the most appropriate pH environment for aprolonged time period due to delayed gastric emptying. In someinstances, the controlled-release oral liquid dosage form includesaqueous solutions or suspensions, partially aqueous solutions orsuspensions such as, for example, high or low molecular weight glycerin,alcohols and glycols or even non-aqueous solutions or suspensions suchas, for example, high or low molecular weight glycerin, alcohols andglycols.

In some instances, a liquid composition disclosed herein further cancomprise one or more excipients: acorbic acid, EDTA dihydrate,glycerine, citric acid monohydrate, sodium citrate dihydrate, sorbitolsolution (e.g., 70%), sucralose, food color (e.g., FD&C Yellow #6), foodor fruit flavor (e.g., artificial or natural orange flavor), mint flavor(e.g., artificial or natural peppermint flavor), or water. In someinstances, the liquid composition further can comprise one or both ofsodium benzoate and sodium proionate. In some instances, the liquidcomposition further can comprise one or more of propylparaben,methylparaben, or propylene glycol. In some instances, the liquidcomposition further can comprise pseudoephedrine or a pharmaceuticallyacceptable salt thereof (e.g., pseudoephedrin HCl).

Methods of Making Liquid Compositions

In one aspect, a method for preparing a liquid composition disclosedherein includes blending one or more beads having one or more activeagents with a dense, thixotropic solution having a density that can beat or about the density of the one or more beads and a thixotropicagent, water and one or more preservatives under conditions that reducebubble formation. In another aspect, a method for preparing a liquidcomposition includes blending a mixture comprising one or more beadscomprising one or more active agents, a thickening agent and asurfactant by mixing with a low cavitation propeller and recirculatingthe mixture under the surface of the mixture so as to minimize bubbleformation. In another aspect, a method for preparing a liquidcomposition includes blending a mixture comprising one or more activeagents on or about a carrier a thickening agent under conditions thatminimize the introduction of air. The conditions that minimize, reduceand/or eliminate the introduction of air and/or air bubbles include oneor more of the following steps used alone, in combination and/or in anyorder: using a diaphragm pump to combine, e.g., the water and thethixotropic agent and one or more preservatives, colorants andflavorants; placing the recirculating tube below the surface of theliquid; adding liquids along the side of a vessel holding the liquid;sprinkling beads (e.g., one or more beads that includes one or moreactive agents) onto the surface of the liquid; mixing the solution inthe absence of one or more paddles that scrape the vessel; mixing thesolution with a propeller mixer; mixing the solution with a propellermixer at a speed that reduces or minimizes cavitation and combinationsof two or more of these steps. In another aspect, a method for preparinga liquid composition includes blending a mixture of one or morecontrolled-release beads with one or more active agents on a carrier ina solution having a low ionic concentration and a thixotropic agent,under conditions that minimize the introduction of air bubbles.

As used herein, the term “thixotropic” can be used to describe one ormore agents, e.g., certain gels, which liquefy when subjected tovibratory forces like simple shaking, and then solidify again when leftstanding. Thixotropic behavior can be observed when long-chain moleculestend to orient themselves in the direction of flow; as the applied forcecan be increased, the resistance to flow can be decreased. Yet when highshear stress can be removed, the solution will quickly revert to itsoriginal viscous state. Some celluloses exhibit thixotropic behaviorwherein the solution returns to its viscous state over a period of time.Examples of thixotropic agents include cellulose (e.g.,carboxymethylcellulose), gums (e.g., xanthan), collagen, gelatin, andaerogels.

In some instances, when formulated with particles, e.g., microparticlesor nanoparticles, the release profile of one or more active agents areeasily be adapted by adding a coating, e.g., a hard or soft gelatincoating, a starch coating, a resin or polymer coating and/or acellulosic coating. Although not limited to microparticles ornanoparticles (as in, e.g., microcapsules or nanocapsules), such dosageforms can be further coated with, for example, a seal coating, anenteric coating, an extended release coating, or a targeted delayedrelease coating. A coating can be applied to an active that can becompressed, molded or extruded. A coating can be applied through anaqueous dispersion or after dissolving in appropriate solvent.

In some instances, a carrier disclosed herein can be fully or partiallybiodegradable. Exemplary carriers include permeable and semipermeablematrices or polymers that control the release characteristics of theformulation. Such polymers include, for example, cellulose acylates, andacetates, as well as the selectively permeable polymers formed by thecoprecipitation of a polycation and a polyanioni. Other carriersinclude, e.g., starch, modified starch, and starch derivatives, gums,including but not limited to xanthan gum, alginic acid, other alginates,benitoniite, veegum, agar, guar, locust bean gum, gum arabic, quincepsyllium, flax seed, okra gum, arabinoglactin, pectin, tragacanth,scleroglucan, dextran, amylose, amylopectin, dextrin, etc., cross-linkedpolyvinylpyrrolidone, ion-exchange resins, such as potassiumpolymethacrylate, carrageenan (and derivatives), gum karaya,biosynthetic gum, etc. Other useful polymers include: polycarbonates(linear polyesters of carbonic acid); microporous materials (bisphenol,a microporous poly(vinylchloride), micro-porous polyamides, microporousmodacrylic copolymers, microporous styrene-acrylic and its copolymers);porous polysulfones, halogenated poly(vinylidene), polychloroethers,acetal polymers, polyesters prepared by esterification of a dicarboxylicacid or anhydride with an alkylene polyol, poly(alkylenesulfides),phenolics, polyesters, asymmetric porous polymers, cross-linked olefinpolymers, hydrophilic microporous homopolymers, copolymers orinterpolymers having a reduced bulk density, and other similarmaterials, poly(urethane), cross-linked chain-extended poly(urethane),poly(imides), poly(benzimidazoles), collodion, regenerated proteins,semi-solid cross-linked poly(vinylpyrrolidone). Additional additives andtheir levels, and selection of a primary coating material or materialswill depend on the following properties: resistance to dissolution anddisintegration in the stomach; impermeability to gastric fluids anddrug/carrier/enzyme while in the stomach; ability to dissolve ordisintegrate rapidly at the target intestine site; physical and chemicalstability during storage; non-toxicity; easy application as a coating(substrate friendly); and economical practicality.

Treatments and Uses

In some cases, the present disclosure provides a method of inhibiting orkilling one or more bacteria, comprising contacting the antibioticcomposition disclosed herein with the one or more bacteria. In somecases, the present disclosure provides a method of treating a bacterialinfection, comprising contacting the antibiotic composition disclosedherein with the bacterial infection. In some instances, the one or morebacteria comprise one or more Gram-negative bacteria. In some instances,the one or more bacteria comprise one or more multidrug-resistanceGram-negative bacteria. In some instances, the one or more bacteria cancomprise K. pneumonia, A. baumannii, P. aeruginosa, E. cloacae, E. coli,or any combination thereof. In some instances, the bacterial infectionor one or more bacteria can be on a surface. In some instances, thebacterial infection or one or more bacteria can be in a mammal. In someinstances, the bacterial infection or one or more bacteria can be in ahuman. In some instances, the contacting can be by injection, forexample intravenous or subcutaneous injection. In some instances, thecontacting can be by topical application. In some instances, thecontacting can be by mouth. In some instances, the contacting lasts forat least about: 1 minute, 2 minute, 3 minutes, 4 minutes, 5 minutes, 10minutes, 20 minutes, 30 minute, 40 minutes, 50 minutes, 1 hour, 2 hours,3 hours, 4 hours, 5 hours, 6 hours, 7 hour, 8 hours, 9 hours, 10 hours,11 hours, 12 hours, 18 hours, one day, two days, three days, four days,five days, six days, one week, or one month. In some instances, thecontacting occurs 1, 2, 3, 4, 5, 6, 7, or 8 times hourly or daily. Insome instances, the contacting occurs about every 3, 4, 5, 6, 7, 8, 9,10, 11, or 12 minutes or hours daily. In some instances, the antibioticcomposition can be in a single unit dose. In some instances, the amountof the organoselenium agent contacted with the bacterial infection orone or more bacteria can be about 10 to 100 mg, about 10 to 50 mg, orabout 20 to 30 mg, for example about 25 mg, per dosage. In someinstances, an amount of the silver contacted with the bacterialinfection or one or more bacteria can be about 1 to 20 mg, about 1 to 10mg, or about 5 to 7 mg, for example about 6 mg, per dosage.

In some instances, an antibiotic composition disclosed herein can beused to treat or prevent bacterial infections or viral infection, and insome instances, protozoan infections. In some instances, the treatmentinhibits or kills one or more bacteria. In some instances, theantibiotic composition can be given as a preventive measure(prophylactic) to at-risk populations such as those with a weakenedimmune system (particularly in HIV cases to prevent pneumonia), thosetaking immunosuppressive drugs, cancer patients and those havingsurgery. In some instances, the antibiotic composition can be used insurgical procedures to help prevent infection of incisions made. In someinstances, the antibiotic composition can be used in dental antibioticprophylaxis to prevent bacteremia and consequent infective endocarditis.In some instances, the antibiotic composition can be used to preventinfection in cases of neutropenia particularly cancer-related.

In some instances, the antibiotic composition can be applied orally,topically, or by injection, e.g., intravenously, subcutaneously, orintramuscularly. In some instances, the antibiotic composition can begiven topically, for example for some skin conditions including acne andcellulitis, or in the form of eye drops onto the conjunctiva forconjunctivitis or ear drops for ear infections and acute cases ofswimmer's ear. In some stances, advantages of topical applicationinclude achieving high and sustained concentration of antibiotic at thesite of infection; reducing the potential for systemic absorption andtoxicity, and total volumes of antibiotic required are reduced, therebyalso reducing the risk of antibiotic misuse. In some instances, theantibiotic composition can be applied topically over certain types ofsurgical wounds to reduce the risk of surgical site infections.

In some instances, an antibiotic composition disclosed herein, such asin a form of coating, are used to sterilize a surface. For example, theantibiotic composition can be applied to surgical equipment, and anysurface in contact with surgical equipment, prior to an operation.Scientific equipment can also be coated with such antibiotic compositionto prevent cross contamination of certain microbes that could interferewith a measurement to be taken with the equipment. In some cases, theantibiotic composition can be applied in the form of a film, sheet,liquid, aerosol, or coating to a biological or non biological surface.Further applications can include adhering an antibiotic composition ontoa transplanted organ to prevent infection by a pathogen during atransplant process.

In some instances, a subject suitable for the treatment can be asurface. In some instances, a subject suitable for the treatment can bea mammal, e.g., a human, mouse, rat, guinea pig, dog, cat, horse, cow,pig, or non-human primate, such as a monkey, chimpanzee or baboon. Insome instances, the subject can be a human. In some instances, thesubject can be an adult. In some instances, the subject can be a child.In some instances, the subject can be 2 years of age or older, 4 yearsof age or older, 6 years of age or older, 12 years of age or older, or18 years of age or older. In some instances, a subject suitable for thetreatment can be younger than 18 years of age, 12 years of age, or 6years of age.

In some instances, compositions disclosed herein are administered to asubject at about every 4 to about 6 hours, about every 12 hours, aboutevery 24 hours, about every 48 hours, or more often. In some instances,a composition disclosed herein can be administered once, twice, threetimes, four times, five times, six times, seven times, eight times, ormore often daily. In some instances, a dosage form disclosed hereinprovides an effective plasma concentration of an active agent at fromabout 1 minute to about 20 minutes after administration, such as about:2 min, 3 min, 4 min, 5 min, 6 min, 7 min, 8 min, 9 min, 10 min, 11 min,12 min, 13 min, 14 min, 15 min, 16 min, 17 min, 18 min, 19 min, 20 min,21 min, 22 min, 23 min, 24 min, 25 min. In some instances, a dosage formof the disclosure herein provides an effective plasma concentration ofan active agent at from about 20 minutes to about 24 hours afteradministration, such as about 20 minutes, 30 minutes, 40 minutes, 50minutes, 1 hr, 1.2 hrs, 1.4 hrs, 1.6 hrs, 1.8 hrs, 2 hrs, 2.2 hrs, 2.4hrs, 2.6 hrs, 2.8 hrs, 3 hrs, 3.2 hrs, 3.4 hrs, 3.6 hrs, 3.8 hrs, 4 hrs,5 hrs, 6 hrs, 7 hrs, 8 hrs, 9 hrs, 10 hrs, 11 hrs, 12 hrs, 13 hrs, 14hrs, 15 hrs, 16 hrs, 17 hrs, 18 hrs, 19 hrs, 20 hrs, 21 hrs, 22 hrs, 23hrs, or 24 hrs following administration. In some instances, the activeagent can be present in an effective plasma concentration in a subjectfor about 4 to about 6 hours, about 12 hours, about 24 hour, or 1 day to30 days, including but not limited to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29or 30 days.

In some instances, an antibiotic composition disclosed herein inhibitsor kills one or more multidrug-resistant bacteria. In some instances, anantibiotic composition disclosed herein inhibits or kills one or moreGram-positive bacteria or Gram-negative bacteria. In some instances, anantibiotic composition disclosed herein inhibits or kills one or moremultidrug-resistant Gram-positive bacteria. In some instances, anantibiotic composition disclosed herein inhibits or kills one or moremultidrug-resistant Gram-negative bacteria, which have nonsusceptibilityto at least one agent in three or more antimicrobial categories. In someinstances, an antibiotic composition disclosed herein inhibits or killsone or more bacteria, including Bacillus cereus var mycoides,Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus,Streptococcus faecalis, Aspergillus niger, Aureobasiduim pullulans,Chaetomium globosum, Gliocladium virens, Penicillum funiculosum, Candidaalbicans, Acinetobacter baumannii, Enterobacteriaceae, cocci, bacilli,vancomycin-resistant enterococci, or Saccharomyces cerevisiae. In someinstances, an antibiotic composition disclosed herein inhibits or killsone or more Gram-negative bacteria, including K. pneumonia, A.baumannii, P. aeruginosa, E. cloacae, E. coli, or any combinationthereof. In some instances, an antibiotic composition disclosed hereininhibits or kills one or more of Escherichia coli (E. coli), Salmonella,Shigella, and other Enterobacteriaceae, Pseudomonas, Moraxella,Helicobacter, Stenotrophomonas, Bdellovibrio, acetic acid bacteria,Legionella, cyanobacteria, spirochaetes, green sulfur, and greennon-sulfur bacteria. In some instances, the antibiotic compositiondisclosed herein exhibits a 50% inhibitory concentration (IC50) of lessthan about: 50 μM, 25 μM, 20 μM, 10 μM, 5 μM, 1 μM, 0.5 μM, 0.1 μM, 50nM, 25 nM, 20 nM, 10 nM, 5 nM, or 1 nM to one or more bacteria.

In some instances, an antibiotic composition disclosed herein treatsinfection caused by one or more organism(s) that are species ofStaphylococcus (e.g., Staphylococcus aureus, Staphylococcusepidermidis), Streptococcus (e.g., Streptococcus viridans, Streptococcuspneumoniae), Enterococcus, Bacillus, Corynebacterium, Propionibacterium,Chlamydia, Moraxella, Haemophilus and Neisseria. In some instances, thespecies are Staphylococcus aureus, Staphylococcus epidermidis,Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcusviridans, Enterococcus faecalis, Corynebacterium sp., Propionibacteriumsp., Moraxella catarrhalis and Haemophilus influenzae.

In some instances, an antibiotic composition disclosed herein can besuitable for topical administration to an eye, such as a form of eyedrop or eye cream, the composition comprising: a combination of activeagents in a concentration effective for treatment and/or prophylaxis ofa gram-positive or gram-negative bacterial infection of at least onetissue of the eye, and at least one ophthalmically acceptable excipientthat reduces a rate of removal of the composition from the eye bylacrimation.

In some instances, an antibiotic composition disclosed herein penetratesor dissolves a microbial biofilm. A microbial biofilm, also referred toas a biological biofilm, can be a community of microbial cells embeddedin an extracellular matrix of polymeric substances and adherent to abiological or a non-biotic surface. A range of microorganisms (bacteria,fungi, and/or protozoa, with associated bacteriophages and otherviruses) can be found in these biofilms. Biofilms are ubiquitous innature, are commonly found in a wide range of environments. Biofilms arebeing increasingly recognized by the scientific and medical community asbeing implicated in many infections, and especially their contributionto the recalcitrance of infection treatment. Biofilms can be etiologicagents for a number of disease states in mammals and are involved in 80%of infections in humans. Examples can include skin and wound infections,middle-ear infections, gastrointestinal tract infections, peritonealmembrane infections, urogenital tract infections, oral soft tissueinfections, formation of dental plaque, eye infections (includingcontact lens contamination), endocarditis, infections in cysticfibrosis, and infections of indwelling medical devices such as jointprostheses, dental implants, catheters and cardiac implants. Microbes inbiofilms can be significantly more resistant to antimicrobial treatmentthan their planktonic counterparts. Biofilm formation is not limitedsolely to the ability of microbes to attach to a surface. Microbesgrowing in a biofilm can interact more between each other than with theactual physical substratum on which the biofilm initially developed.

EXAMPLES Example 1. In Vitro Experiments

1.1 Results

Combination of Silver with Ebselen Exhibited Selective SynergisticToxicity Against Bacteria

The effect of silver nitrate with ebselen in combination on the growthof Gram-negative model bacteria, E. coli, was investigated in themicroplates. DHB4 overnight cultures were diluted 1:1000 times inLuria-Bertani (LB) medium, and treated with ionic silver (Ag⁺) as anitrate salt (AgNO3) for 16 h. Ag⁺ alone inhibited E. coli growth with aminimal inhibition concentration (MIC) of 42 μM after 16 h treatment,while the addition of 2 μM ebselen dramatically decreased the MIC of Ag⁺to 4.2 μM (p=0.000028<0.001) (FIG. 1A). Meanwhile, 5 μM Ag⁺ and 2.5 μMebselen in combination showed no synergistic toxicity on human HeLacells (p=0.98>0.05) (FIG. 1B). In addition, the toxicity of ebselenitself (2, 4, 8 μM) on bacterial and mammalian cells was similar (FIGS.1A and 1B) with no effects on bacterial growth (FIG. 1C). These resultsindicate that treatment of Ag⁺ with ebselen in combination exhibitssignificant selective synergistic toxicity on bacteria over mammaliancells, and the dramatic decrease of MIC of silver against bacteria inthe presence of ebselen make the systemic medical use of silverfeasible.

The large scale growth inhibition of E. coli by Ag⁺ with ebselen incombination was also observed in shaking testing 15 ml tubes. E. coliDHB4 cells were grown until an OD_(600 nm) of 0.4, and treated with 5 μMAg⁺ and serial concentrations of ebselen (0, 20, 40, 80 μM). The growthcurves showed a synergistic bacteriostatic effect of Ag+ with ebselen incombination in LB medium (FIG. 2A), and the synergistic bactericidaleffect of 5 μM Ag+ and 80 μM ebselen in combination was furtherconfirmed by the colony formation assay on LB-agar plates (FIG. 2B).Meanwhile, only 80 μM ebselen itself could inhibit E. coli growth infirst 8 h, and gains back into normal 12 h post-treatment. While 40 μMebselen or 5 μM Ag⁺ alone did not inhibit bacterial growth, Ag⁺ withebselen in combination resulted in strong inhibition of E. coli growth(FIGS. 2A and 2B). In line with this, 5 μM Ag+ and 20 μM ebselen incombination enhanced the frequency of propidium iodide (PI) staining(p=0.00083<0.001) (FIGS. 2C and 2D). PI is a membrane-impermeablefluorescent dye that has been widely used to detect permeation of cellmembrane and dead cells. Above all, these results indicate that Ag⁺ andebselen in combination exhibited a selective synergistic effect onbacteria.

Clinically Isolated Five Most Difficult-to-Treat MDR Gram-NegativePathogens were Sensitive to Ag⁺ with Ebselen in Combination

There are five most difficult-to-treat MDR Gram-negative pathogenspecies in the clinic, which are also typical GSH-positive bacteria:Klebsiella pneumonia, Acinetobacter baumannii, Pseudomonas aeruginosa,Enterobacter cloacae and Escherichia coli. Two strains of each specieswere isolated, overnight cultures were diluted 1:1000 times in LBmedium, and treated with Ag⁺ with a serial concentration of ebselen incombination for 16 h. The synergistic bactericidal effects of Ag⁺ withebselen in combination against all 10 tested strains were observed(Table 1). Among these five species, A. baumannii and E. cloacae arevery readily formed drug-resistant strains, which are needed to betreated by or the fourth-generation cephalosporin in the clinic,including imipenem, cefepime, and cefotaxime, etc. The isolatedimipenem, cefepime, and cefotaxime-resistant A. baumannii (AB-1/2) andE. cloacae (ECL-1) strains were identified (Table 2, and 3), and weresensitive to Ag⁺ with ebselen in combination (Table 1). These resultsindicate that Ag⁺ with ebselen in combination might be ‘the lastlife-saving straw’ that are active against a range of bacteria withexisting resistance, which would increase the correct chance forempirically-prescribed therapy, even for infections resistant toconventional antibiotics.

Ag⁺ with Ebselen in Combination Directly Disrupted Bacterial Trx and GSHSystems

Since Ag⁺ and ebselen are generally thought to be thiol-targetingagents, bacterial TrxR or Trx activity in cells were treated by Ag⁺ withebselen in combination (FIGS. 3A and 3B). While TrxR and Trx activitiesin cell extracts were not influenced by either Ag⁺ or ebselen alone, 5μM Ag⁺ and 20 μM ebselen in combination resulted in a dramatic loss ofTrxR (p=0.00018<0.001) and Trx (p=0.0036<0.01) activities (FIGS. 3A and3B). Consistent with this observation, the redox state of Trx1 measuredby Redox Western Blot was also affected by treatment of Ag⁺ with ebselenin combination. Trx1 was mostly in reduced form in untreated bacteria,which became oxidized upon treatment of drugs in combination (FIG. 3C).A Trx2 antibody was used to detect oxidized Trx2 in the experiment toinvestigate the effect of treatment on the redox state of Trxs. ReducedTrx2 could not be detected by this antibody probably because of theblockage of the recognition site. None of the oxidized Trx2 was observedupon the treatment, while the positive control diamide-oxidized Trx2 wasdetected (FIG. 3D). These results showed that Trx2 was less sensitive tothe treatment compared to Trx1. In addition, the protein levels of Trx 1and 2 were not affected by the 10 min treatment with Ag⁺ and ebselencombination (FIGS. 3C and 3D).

The addition of 5 μM Ag⁺ and 20, 40 or 80 μM ebselen also decreased theGSH levels. Five μM Ag⁺ and 20 μM ebselen in combination treatmentdepleted the functional GSH in 10 min compared with control(p=0.000021<0.001) (FIG. 3E). Ebselen alone at 80 and 40 μM also reducedGSH levels, albeit less efficiently than the corresponding drugs incombination at the same concentration (p=0.000076, and 0.000029, <0.01).Instead, neither 5 μM Ag⁺ nor 20 μM ebselen modulated GSH levels in theconditions tested compared with control (p=0.081, and 0.712, >0.05)(FIG. 3E).

Whether Ag⁺ with ebselen in combination decreased or depleted GSH couldaffect proteins S-glutathionylation was further explored (FIG. 3F).Proteins S-glutathionylation was decreased in Ag⁺ with ebselen treatedbacteria, but not in those incubated only with 5 μM Ag⁺ or 20 μM ebselenalone. Thus the presence of 5 μM Ag⁺ decreased proteinS-glutathionylation when combined with 20 μM ebselen reflecting the lossof GSH (FIG. 3F).

Since Trx and GSH/Grx are major thiol-dependent systems, investigatedwere the effects of Ag⁺ with ebselen in combination on Trx or GSHsystem-deficient E. coli redox mutants. E. coli mutants lacking GSHsystem components (gshA⁻) and living on Trx and TrxR were more sensitiveto Ag⁺ and ebselen treatment compared with the wild type (WT) (Table4-6). All results showed that Ag⁺ with ebselen in combination has strongsynergistic effects on bacterial Trx and GSH systems, and targetingthiol-dependent systems as a novel antibiotic strategy.

Silver Irreversibly Inhibits Bacterial Trx and TrxR Activities

Cellular TrxR and Trx1 enzyme activities were decreased while thecorresponding protein levels were unaltered by the treatment of Ag⁺ withebselen in combination, suggesting that TrxR and Trx were inhibited.Since ebselen is a known reversible competitive inhibitor of bacterialTrxR, the effect of Ag⁺ on the activity of E. coli TrxR and Trx wasinvestigated. When 100 nM of NADPH-pre-incubated E. coli TrxR wasincubated with Ag⁺, the IC₅₀ was about 50 nM (FIG. 4A), similar to thegold compound auranofin. To detect whether E. coli TrxR can bespecifically inhibited by Ag⁺, the enzyme was incubated with Ag⁺ in thepresence of reduced E. coli Trx1, the inhibitory efficiency toward TrxRdecreased (FIG. 4A). This indicated that Trx also reacted with Ag⁺ andplayed a protective role for the TrxR. Fluorescence spectroscopy furtherverified that Ag⁺ interacted with reduced Trx1 and changed itsfluorescence spectra (FIG. 4B). Incubation with 1-10 μM Ag⁺ increasedthe tryptophan fluorescence intensity of 10 μM Trx1. Meanwhile, thefluorescent intensity of 10 μM Trx1 decreased when treated with 20-100μM Ag⁺ (FIG. 4B). In line with this, the activity of Trx decreased alongwith the increase of Ag⁺ concentration (FIG. 4C). The inhibition of Trx1by Ag⁺ was irreversible since the Trx1 activity was not recovered afterdesalting (p=0.00021<0.001) (FIG. 4D). This indicated that Ag⁺ formed atight complex with the sulfhydryl groups in reduced E. coli Trx1. Allthese results show that silver irreversibly inhibits bacterial Trx andTrxR activities.

ROS is a Determining Factor for Synergistic Bactericidal Effect of Ag⁺and Ebselen

One major function of GSH and Trx systems is to scavenge ROS to keepcellular redox balance and protect against oxidative stress. Theinhibition of the Trx system and depletion of GSH may responsible forthe elevation of ROS. To determine whether increased ROS levelsaccounted for the bactericidal effect, ROS levels were determined in Ag⁺and ebselen treated cells. Treatment with either 5 μM Ag⁺ or 20 μMebselen alone did not change ROS concentrations, while the combinationof 5 μM Ag⁺ and 20 μM ebselen resulted in increased levels of ROS(p=0.00012<0.001) (FIGS. 5A and 5B). Further, the enhancement of H₂O₂levels caused by the treatment with 5 μM Ag⁺ and 20 μM ebselen incombination was also verified by Amplex Red method(p=0.00057<0.0001)(FIG. 5C). In addition, E. coli mutants lacking OxyRcomponents (OxyR⁻) that impair E. coli dehydratase clusters from H₂O₂injury were more sensitive to Ag⁺ and ebselen treatment compared withthe wild type (WT) (Table 4-6). All results showed that lethality of Ag⁺with ebselen against bacteria is accompanied by ROS generation.

1.2 Materials and Methods

Bacterial Strains

All in vitro experiments were performed with Escherichia coli (E. coli)DHB4 and its derived redox phenotypes (Table 6), and clinically isolatedmultidrug-resistance (MDR) Gram-negative strains (Table 2, 3, 7). All invivo experiments were performed with E. coli ZY-1 (Table 7), which wasisolated from the urine of clinical patient in the First AffiliatedHospital of Three Gorges University in Hubei Province, P. R. China, withan approval for research from the Ethics Committee of First AffiliatedHospital of Three Gorges University and an informed-consent of thepatient. The strain was thoroughly identified and stored. Other clinicalisolated MDR Gram-negative strains (Table 2, 3) were obtained fromclinical patients in Renmin Hospital of Three Gorges University in HubeiProvince, PRC, with all approvals and informed consents.

Antibiotics and Chemicals

All experiments were performed in Luria-Bertani (LB) medium (EMDmillipore). Unless otherwise specified, the following concentrationswere used for the antibacterial experiments with E. coli strains andclinical pathogens: 0, 1, 2, 4, 5, 20, 40, 80 μM 2-Phenyl-1,2-benzisoselenazol-3(2H)-one (ebselen) (Daiichi), and 0, 0.625, 1.25,2.5, 5, 10, 20, 40, 80 μM silver nitrate (Sigma-Aldrich).4-acetamido-4′-maleimidylstilbene-2,2′-disulfonic acid (AMS)(Invitrogen), protease inhibitor cocktails (Roche), DC™ protein assay(Bio-RAD), propidium iodide (PI) (BD Biosciences), E. coli DHB4 TrxR,sheep anti-E. coli Trx1 antibody and Rabbit anti-E. coli Trx2 antibodywere from IMCO Corp. (Stockholm, Sweden), Goat anti-Rabbit IgG-HRP(Santa Cruz, lot #H1015), Rabbit anti-Goat IgG-HRP (Southern Biotech,lot #12011-PG56), IgG2a mouse monoclonal antibody forglutathione-protein complexes (VIROGEN, lot #101-A, clone number D8),4-12% bolt Bis-Tris gel (VWR), all the other reagents were fromSigma-Aldrich.

Synergistic Effect of Silver with Ebselen in Combination on E. coliGrowth

E. coli DHB4 cells overnight cultures were diluted 1:1000 times inLuria-Bertani (LB) medium and treated with serial concentrations ofAgNO3 and/or ebselen for 16 h. The cell viability was determined bymeasuring the absorbance at 600 nm. The culture treated with 0.8% (v/v)DMSO was used as a control.

Toxicity Analysis of Silver with Ebselen in Combination AgainstMammalian Cells

HeLa cells were purchased from ATCC, and through mycoplasma detectionand human cell line authentication by STR analysis (ATCC, U.S.A). HeLacells cultured in DMEM medium supplemented with 10% FCS, 100 units/mlpenicillin, and 100 μg/ml streptomycin at 37° C. in a 5% CO₂ incubator.The cells were seeded in 96 micro-well plates and grown till 70-80%confluency. The cells were treated with serial combinations of ebselenand AgNO3 for 24 h. The cell toxicity was detected by MTT assay.

Antibacterial Effect of Silver with Ebselen in Synergistic Combinationon the Growth of Clinical Isolated MDR Gram-Negative Strains

Ten clinical isolated MDR Gram-negative (GSH-positive) strains weregrown until an OD_(600 nm) of 0.4 and were diluted 1:100 into 100 μl ofLB medium in 96 micro-well plates. Serial dilutions of 100 μl ebselenand AgNO3 were added to the individual wells. The minimum inhibitoryconcentration (MIC) was determined after 16 h culture at 37° C. Theculture treated with 0.8% (v/v) DMSO was used as a control.

Detection of Bactericidal Effect of Silver with Ebselen in Combinationon E. coli Strains

E. coli DHB4 cells were grown in 15 ml tubes until an OD_(600 nm) of0.4, and treated with 5 μM AgNO3 and serial concentration of ebselen (0,20, 40, 80 μM) in combination. The survival of untreated E. coli wascompared with the antibiotic-treated cells by measuring OD_(600 nm) andcounting the colonies. For colony formation assay, cells were harvestedat 10 min, 1 h, 2 h, and 4 h by centrifugation at 6,000 rpm for 5 minand thoroughly washed 3 times with PBS. The cells were serially dilutedin PBS, and 100 μl cultures were plated on LB plates. The colonies werecounted after overnight incubation, and CFU/ml was calculated using thefollowing formula: [(colonies)*(dilution factor)]/(amount plated).

Further, cells cultured and washed as above were harvested at 10 min bycentrifugation at 6,000 rpm for 5 min and thoroughly washed 3 times withPBS. Nuclei were stained with 5 μg/ml propidium iodide (PI) for 20 minin the absence of a cell permeate and analyzed by flow cytometry (CyAnadp, Beckman coulter).

Measurement of Trx/TrxR Activity and GSH Amount in Silver and EbselenTreated E. coli Cell Lysates

E. coli DHB4 cells were grown till the absorbance at OD_(600 nm) of 0.4in LB medium, and the bacterial cells were treated with differentdilutions of ebselen and AgNO3 for 10 min. Cells were harvested bycentrifugation at 6,000 rpm for 5 min and thoroughly washed 3 times withPBS, then cells were re-suspended in lysis buffer (25 mM Tris·HCl, pH7.5, 100 mM NaCl, 2.5 mM EDTA, 2.5 mM EGTA, 20 mM NaF, 1 mM Na₃VO₄, 20mM sodium ß-glycerophosphate, 10 mM sodium pyrophosphate, 0.5% TritonX-100) containing protease inhibitor cocktail and lysed by sonication.The cell lysates were obtained by centrifugation at 13,000 rpm for 20min and the protein concentration was measured by Lowry protein assay(Bio-Rad DC™).

E. coli DHB4 TrxR activity in cell extracts was measured by a DTNBreduction activity assay. The experiments were performed with 96micro-well plates in the solution containing 50 mM Tris·HCl (pH 7.5),200 μM NADPH, 1 mM EDTA, 1 mM DTNB, in the presence of 5 μM E. coli Trx.The absorbance at 412 nm was measured for 5 min with a VERSA micro-wellplate reader and the slope of initial 2 min was used to represent TrxRactivity. The Trx activity was detected by this method coupled with 100nM E. coli TrxR instead of 5 μM E. coli Trx in the reaction mixture.

To measure GSH levels, 25 μg of the cell lysates was added in thesolution containing 50 nM GR, 50 mM Tris·HCl (pH 7.5), 200 μM NADPH, 1mM EDTA, 1 mM DTNB. The absorbance at 412 nm was measured for 5 min.

Trx Redox State in E. coli Treated with Silver and Ebselen inCombination

E. coli DHB4 cells were grown till the absorbance at OD_(600 nm) of 0.4in LB medium, and the bacterial cells were treated with differentdilutions of ebselen and AgNO3 for 10 min. Western blotting wasperformed to detect the Trx1 and Trx2 redox state of the ebselen andAgNO3 treated E. coli cells. The cells were harvested by centrifugationat 6,000 rpm for 5 min and thoroughly washed 3 times with PBS, andprecipitated the protein with 5% TCA in 1.0 ml. The precipitates werewashed with 1 ml pre-ice-cold acetone for 3 times, and dissolved in 50mM Tris·HCl (pH 8.5) with 0.5% SDS containing 15 mM AMS at 37° C. for 2h. Proteins were obtained by centrifugation at 13,000 rpm for 20 min toremove the pellets, and the protein concentration was measured by Lowryprotein assay (Bio-Rad DC™). Redox state of Trx1 and Trx2 was detectedwith sheep anti-E. coli Trx1 antibody and (Rabbit anti-E. coli Trx2antibody) at 1:1000 dilution, followed by the detection ofChemiluminescence Reagent Plus.

Proteins S-Glutathionylation in E. coli Treated with Silver and Ebselenin Combination

Total proteins S-glutathionylation of the ebselen with AgNO3 incombination treated E. coli cells were detected by Western blotting.Cells were cultured and washed as described above, and re-suspended inlysis buffer (25 mM Tris·HCl, pH 7.5, 100 mM NaCl, 2.5 mM EDTA, 2.5 mMEGTA, 20 mM NaF, 1 mM Na₃VO₄, 20 mM sodium ß-glycerophosphate, 10 mMsodium pyrophosphate, 0.5% Triton X-100, protease inhibitor cocktail)containing 30 mM IAM. After lysed by sonication, the cell lysates wereobtained by centrifugation at 13,000 rpm for 20 min. Proteinconcentration was measured by Lowry protein assay (Bio-Rad DC™). Sampleswere incubated with SDS-loading buffer at 90° C. for 10 min, and thenseparated on the 4-12% bolt Bis-Tris gel with MES running buffer (150V,40 min). Western blotting assay was performed with IgG2a mousemonoclonal antibody (VIROGEN, 101-A/D8) for glutathione-proteincomplexes.

Synergistic Effect of Silver and Ebselen on the Growth of E. coli DHB4Redox Phenotypes

Eleven E. coli DHB4 redox phenotypes were grown until an OD_(600 nm) of0.4, and were diluted 1:100 into 100 μl of LB medium in 96 micro-wellplates. Serial dilutions of ebselen and AgNO3 were added to theindividual wells. The minimum inhibitory concentration (MIC) wasdetermined after 24 h culture at 37° C. The culture treated with 0.8%(v/v) DMSO was used as a control.

Inhibition of Recombinant Bacterial Trx/TrxR by Silver

Inhibition of recombinant bacterial TrxR by Silver was performed byusing E. coli enzyme. The experiments were performed with 96 micro-wellplates in the solution containing 50 mM Tris·HCl (pH 7.5), 200 μM NADPH,1 mM EDTA, 1 mM DTNB, in the presence of 5 μM E. coli Trx. Theabsorbance at 412 nm was measured for 5 min with a VERSA micro-wellplate reader and the slope of initial 2 min was used to represent TrxRactivity. The Trx activity was detected by this method, coupled with theuse of 100 nM E. coli TrxR instead of 5 μM E. coli Trx in the reactionmixture.

Analysis of Fluorescent Spectra

Fluorescent Spectra of reduced E. coli Trx with Silver were recorded at10 μM in a PerkinElmer Enspire multilabel recorder using an excitationat 280 nm.

Measurement of ROS Production

The E. coli DHB4 cells were grown till the absorbance at OD_(600 nm) of0.4 in LB medium, and the bacterial cells were treated with differentcombinations of ebselen and AgNO3 for 10 min. To analyze the amount ofROS production in the bacteria, cells were harvested by centrifugationat 6,000 rpm for 5 min and thoroughly washed 3 times with PBS, andstained with 5 μM H₂DCF-DA for 20 min. After the incubation, cells werespin down and re-suspended in PBS, and the ROS production was quantifiedby flow cytometry (CyAn adp, Beckman coulter).

H₂O₂ Production

The E. coli DHB4 cells were grown till the absorbance at OD_(600 nm) of0.4 in LB medium, and the bacterial cells were treated with 20 μMebselen and 5 μM AgNO3 for 10 min. Cells were harvested bycentrifugation at 6,000 rpm for 5 min and thoroughly washed 3 times withPBS, and sonicated for 10 s. In the presence of 50 μM Amplex® Redreagent, 0.1 U/mL HRP in 50 mM sodium phosphate buffer, pH 7.4, 50 μlsamples were incubated for 30 minutes at room temperature protected fromlight, and detected with absorbance at 560 nm (Molecular Probes, Eugene,Oreg.).

Effect of Ebselen on E. coli Growth

E. coli DHB4 cells were grown until an OD_(600 nm) of 0.4, and treatedwith serial dilutions of ebselen (0, 2, 4, 8 μM) for 16 h. The cellviability was determined by measuring the absorbance at 600 nm. Theculture treated with 0.8% (v/v) DMSO was used as a control.

Direct Survival Rate Assay

The direct survival rate assay was performed to assess the survivalcapacity of ebselen and AgNO3 treated E. coli DHB4 strain in healthymice blood. The phosphate-buffered saline (PBS, pH 7.6) treated cellswere used as the positive control, and the experiment was performed induplicate. Briefly, blood was extracted from 3 mice and collected inheparinized tubes. Approximately 100 E. coli DHB4 cells were harvestedduring the logarithmic phase, washed with sterile PBS, and added to 100μl blood. After incubation at 37° C. for 6 h, duplicate 100 μl aliquotsfrom each blood sample were spread onto LB agar, and the survivingcolonies were enumerated after overnight incubation. The results showedthat ebselen and AgNO3 could help innate immunity to clear E. coli.

Inhibition of Recombinant Mammalian TrxR by Silver

The experiments were performed with 96 micro-well plates in the solutioncontaining 50 mM Tris·HCl (pH 7.5), 250 μM NADPH, 1 mM EDTA, 1 mM DTNB,in the presence of 10 nM E. coli TrxR. The absorbance at 412 nm wasmeasured for 5 min with a VERSA micro-well plate reader and the slope ofinitial 2 min was used to represent TrxR activity.

Statistical Analysis

Mean, Standard Deviation (SD) and t-test (two tails, unpaired)significances were calculated in GrapPad Prism Software. *: p<0.05, **:p<0.01, ***: p<0.001.

Example 2. In Vivo Experiments

2.1 Results

The bactericidal effect of Ag⁺ with ebselen in combination was alsoobserved in LB medium containing heparinized mice blood. To investigatewhether the bactericidal activity of Ag⁺ with ebselen in combination isalso efficient in vivo, mice were infected i.p. with 6.0×10⁷ or 1.7×10⁶MDR E. coli ZY-1 (Table 7), modeling an acute and a mild peritonitis,respectively. The LD50 of E. coli ZY-1 administered i.p. was 1.3×10⁷CFU/ml. One hour (acute model) or 24 h (mild model) after infection,mice were treated i.p. with ebselen, Ag⁺, or the drugs in combination,or remained untreated. The combination of Ag⁺ and ebselen led to asignificant reduction of bacterial load compared with the control in themild peritonitis model. Mice treated with ebselen alone or leftuntreated showed similar levels of bacteria load after 36 h of infectionwith 6.0×10⁷ E. coli, whereas treatment with Ag⁺ with ebselen incombination achieved a 100-fold reduction compared with control(p=0.0055<0.01)(FIG. 6A). Additionally, 80% of mice treated with Ag⁺with ebselen in combination survived in the acute peritonitis micemodel, compared with 30% in control group (FIG. 6B). These findingsdemonstrated the effective antibacterial effect of Ag⁺ and ebselenagainst MDR Gram-negative pathogen in vivo.

Ag⁺ or ebselen alone has been proven to be safe in previous studies. Totest the toxicity of the combination, mice were divided 5 per group,which were treated with 6 mg AgNO3/kg body weight in combination withserial concentrations of ebselen (10, 15, 20, and 25 mg ebselen/kg bodyweight). Mice were observed for 7 days and remained viable with nomortality. The effect of 25 mg ebselen/kg and 6 mg AgNO₃/kg body weightin combination were evaluated on mice by measuring key metabolite andenzyme concentrations using a Blood Chemistry Analyzer after treatmentfor 6, 24 and 48 hours. The density of lymphocytes and monocytes andsome enzymes, such as alanine transaminase in mice treated with Ag⁺ andebselen in combination were reduced at the initial point (6 h); however,their values gain back into normal at 24 h post-treatment (Table 8),indicating that there is a stress response upon the initial treatment.These results demonstrated that Ag⁺ and ebselen were not toxic for miceat the conditions tested.

2.2 Materials and Methods

Mild Peritonitis Mice Model Assay

Approval from the Medical Animal Care & Welfare Committee of China ThreeGorges University was obtained prior to using the animals for research.Healthy 6-week-old Kunming male mice (body weight, 18±2 g) werepurchased from Laboratory Animal Center of China, Three GorgesUniversity. All mice were kept in individually ventilated cages (fivemice per cage) under a constant dark (12 h)-light (12 h) cycle in aconventional SPF animal house and were free access to food and water.Five mice were sampled randomly to examine bacterial recovery from thebrain, liver, spleen and kidney to rule out E. coli infection beforeexperimental manipulation, and no bacteria were detected.

The experimentation was performed in random block design andsingle-blind trial. The sample size was calculated by power analysis,and estimated as: corrected sample size=sample size/(1−[%attrition/100]). Forty-eight mice were divided into 4 groups, 12mice/group. Inoculation was performed by intraperitoneal injection of100 μl 1.7×10⁶ E. coli ZY-1 cells using a 26-gauge syringe. The inoculumwas delivered in suspension with 8% (w/v) mucin in sterile saline. 24 hafter introduction of the inoculum, 12 mice per group receivedantibacterial treatments. 0, 12, 24, and 36 h post-infection, peritonealwashes were performed by injecting 1.0 ml of sterile saline in theintraperitoneal cavity followed by a massage of the abdomen (100times/mouse). Subsequently, the abdomen was opened and 200 μl ofperitoneal fluid (PF) was recovered from the peritoneum for analysis ofE. coli CFU/ml, and CFU/ml was enumerated. For the CFU/ml measurement,the peritoneal fluid was serially diluted in PBS (pH 7.6). Experimentsare performed triplicate.

Acute Peritonitis Mice Model Assay

The experimentation was designed in random block design and single-blindtrial, and 40 mice were divided into 4 groups, 10 mice/group.Inoculation was performed by intraperitoneal injection of 100 μl of6.0×10⁶ CFU/ml E. coli ZY-1 inoculums using a 26-gauge syringe. Theinoculum was delivered in suspension with 8% (w/v) mucin in sterilesaline. 1 h after introduction of the inoculum, 10 mice per groupreceived antibacterial treatments, and the mice were observed for 7 daysto evaluate overall survival. Experiments are performed duplicate.

In Vivo Toxicity Analysis of Silver with Ebselen in Combination

Five mice per group were treated with 6 mg AgNO3/kg body weight andserial concentration of ebselen (10, 15, 20, 25 mg AgNO₃/kg body weight)intraperitoneally. Mice were observed for 7 days, and the overallsurvival was calculated.

Blood Samples Analysis

Three mice per group were treated with parenterally administered PBS, 25mg ebselen/kg body weight in combination with 6 mg AgNO₃/kg body weight,and vehicle. The animals were observed for two days and retro-orbitalblood sample collection was performed 6, 24 and 48 h after treatment.Blood was collected in heparinized whole blood test tubes and furtheranalyzed by Blood Chemistry Analyzer (SYSMEX XE5000).

Statistical Analysis

Mean, Standard Deviation (SD) and t-test (two tails, unpaired)significances were calculated in GrapPad Prism Software. *: p<0.05, **:p<0.01, ***: p<0.001.

Example 3. Comparison with Conventional Antibiotics

3.1. Results Against E. coli

Combination of Silver with Certain Antibiotics Exhibited SynergisticToxicity Against E. coli

The antibacterial effects of silver nitrate (AgNO₃) and nine antibioticsrepresenting five different functional categories (beta-lactams,aminoglycosides, synthesis, tetracycline, and macrolides) on the growthof a model Gram-negative bacterium, E. coli, was investigated in the 96wells microplates. E. coli DHB4 overnight cultures were diluted 1:1000times in Luria Bertani (LB) medium, and treated with serial dilutions ofionic silver (Ag⁺) as a nitrate salt and 9 antibiotics in combinations,separately, for 24 h. Ebselen was used as the positive control, whichacted synergistically with silver against Gram-negative bacteria. Theresults here showed that 4 (gentamicin, kanamycin, geneticin,tetracycline) out of 9 antibiotics had synergistic activity on E. coliDHB4 growth (Table 9). Further, the Bliss model was used to determinethe nature of the therapeutic effects exhibited by the silver andantibiotics in combinations. The degree of synergy was quantified at 1and 4 h between Ag⁺ and 4 antibiotics (gentamicin, kanamycin, geneticin,and tetracycline) in combinations, and the results showed that Ag⁺ and 4antibiotics indeed had synergistic combinations against E. coli (FIG. 7). All the results pointed out that silver could enhance theantibacterial effects of certain antibiotics against Gram-negativebacteria.

ROS was a Lethal Factor for Synergistic Bactericidal Effects of Ag⁺ andAntibiotics

Ag⁺ and ebselen in combination could induce a high level of ROS, and theeffects of Ag⁺ and antibiotics in combinations need further studies. We,therefore, determined ROS levels in Ag⁺ and 4 antibiotics incombinations treated cells, and Ag⁺ and ebselen in combination was usedas a positive control. The results showed that treatment with 5 μM Ag⁺and 80 μM antibiotics in combinations resulted in increased levels ofROS (p<0.0001) (FIG. 8A). Further, the enhancement of H₂O₂ levels causedby the treatment with 5 μM Ag⁺ and 80 μM antibiotics in combinationswere also verified by Amplex Red method (p<0.0001) (FIG. 8B). Theresults demonstrated that ROS was one of the determining factors forsynergistic bactericidal effects of Ag⁺ and antibiotics in combinationsagainst E. coli.

Ag⁺ and Antibiotics could Disrupt Bacterial Trx System

One major function of thiol-dependent antioxidant systems is to scavengeROS to keep cellular redox balance and protect against oxidative stress.The inhibition of the Trx system and depletion of GSH may responsiblefor the elevation of ROS. Ag⁺ and ebselen in combination has been provento target both bacterial Trx and GSH systems, while the effects of Ag⁺and antibiotics in combinations need further studies. E. coli DHB4 grownto OD_(600 nm) of 0.4 were treated with 5 μM Ag⁺ and 80 μM antibioticsin combinations, and Ag⁺ and ebselen in combination was used as apositive control. Results here showed that after 10 min treatment, theTrx activities in cell extracts treated by Ag⁺ and antibiotics incombinations were dramatically inhibited compared with antibiotics orcontrol group (FIG. 9A, p<0.001); meanwhile, the TrxR activities in cellextracts treated by Ag⁺ and antibiotics in combinations were alsostatistically lowered when compare with antibiotics or control group(FIG. 9B, p<0.05). The same results were obtained when the treatmenttime was prolonged to 60 min. These results suggested that silver andantibiotics in combinations have direct influences on Trx1.

Consistent with this observation, redox state of Trx1 measured by RedoxWestern Blot was also affected by Ag⁺ and antibiotics in combinationsafter 60 min treatment. Trx1 was in reduced form in untreated bacteria,and became oxidized upon the treatment by Ag⁺ and antibiotics incombinations (FIG. 9C). In contrast, only 10 min treatment by Ag⁺ andantibiotics in combinations could not cause Trx1 oxidization. At thesame time, the total protein levels of Trx1 were not affected followinga 10 min or 60 min treatment by Ag⁺ and antibiotics in combinations.These results showed that when targeting Trx system, silver andantibiotics in combinations was not acting as fast as silver and ebselendo. All the results showed that silver and antibiotics in combinationshad direct influences on the Trx system.

Ag⁺ and Conventional Antibiotics could not Directly Disrupt BacterialGSH System

5 μM Ag⁺ and 80 μM ebselen in combination has also been proven todeplete the GSH after 10 min treatment. In that study, only 5 μM Ag⁺ and80 μM gentamicin or kanamycin in combinations could slightly deplete thetotal GSH amount in cell extracts when compared with antibioticsthemselves (p<0.05) (FIG. 10A), meanwhile other combinations showed nodifferences (FIG. 10B) (p>0.05). The same results were obtained when thetreatment time was prolonged to 60 min (FIGS. 11A and 11B). Further, theprotein S-glutathionylation was decreased in Ag⁺ and ebselen incombination treated bacteria, but not in those incubated with 5 μM Ag⁺and antibiotics in combination for 10 min (FIG. 10B) or 60 mintreatments (FIG. 11B).

All the results above suggested that silver and conventional antibioticshad no direct effect on bacterial GSH system when acting againstGram-negative bacteria. 4 out of 9 conventional antibiotics actedsynergistically with silver against E. coli, a model Gram-negativebacterium (Table 9), which might occur through a direct targeting of Trxand TrxR and inducing ROS production (FIGS. 8A-8B, and 9A-9C), but notthe GSH content (FIGS. 10A-10B and 11A-11B). The synergistic effect camealong with the production of reactive oxygen species.

3.2 Results Against Five MDR Gram-Negative Pathogens

The four conventional antibiotics identified in 3.1 were further studiedin comparison with ebselen in combination with silver. Using clinicallyisolated strains of K. pneumonia, A. baumannii, P. aeruginosa, E.cloacae and E. coli, only ebselen at 4 μM, out of five antibioticslowered the MIC of silver dramatically.

Clinically Isolated Five Most Difficult-to-Treat MDR Gram-NegativePathogens were Highly Sensitive to Only Ag⁺ and Ebselen in Combinations

There are five clinically most difficult-to-treat MDR Gram-negativepathogen species: Klebsiella pneumonia, Acinetobacter baumannii,Pseudomonas aeruginosa, Enterobacter cloacae and Escherichia coli. Onestrain from each species was isolated, and overnight cultures werediluted 1:1000 times in LB medium, and treated with serialconcentrations of Ag⁺ and antibiotics in combinations for 24 h (Table10). The results showed that Ag⁺ and antibiotics in combinationexhibited weak antibacterial effects on these MDR Gram-negativebacteria, meanwhile, Ag⁺ and ebselen in combination might be the onlyeffective antibiotic against a range of resistant bacteria. The resultsmight be explained by the fact that silver and antibiotics incombination could only directly disrupt the Trx system but not both theTrx and GSH systems.

Although silver and 4 different antibiotics in combinations coulddirectly inhibit Trx and TrxR, yet the direct effect targeting GSHsystem is not universal. The presence of the GSH-Grx system in E. colimay be regarded as a backup for the Trx system. GSH/Grxs in E. coliparticipate in the antioxidant process by deglutathionylation andtransfer electrons to ribonucleotide reductase. Silver and fourconventional antibiotics in combinations showed no effects on GSHamount, and the S-glutathionylated proteins are not much different fromthat of in control group (FIG. 10 ). Thus, this might explain theanti-MDR-Gram-negative bacteria activities of silver and antibiotics incombinations were much weaker than silver and ebselen in combination.

3.3 Materials and Methods

Bacterial Strains

All in vitro experiments were performed with Escherichia coli (E. coli)DHB4, and clinically isolated multidrug-resistance (MDR) Gram-negativestrains shown below. Clinical isolated MDR Gram-negative strains wereobtained from clinical patients in Renmin Hospital of Three GorgesUniversity in Hubei Province, PRC, with all approvals and informedconsents.

Clinically isolated multidrug-resistant Gram-negative strains used inthis work Strain Description KP-2 K. pneumoniasubsp. pneumonia 0322^(#)AB-1 Acinetobacter baumannii (A. baumannii) H^(#) PA-1 Pseudomonasaeruginosa (P. aeruginose) 1298# ECL-2 E. cloacae 2301# ECO-1Escherichia coli (E. coli) 1139^(#)

Drug sensitivity of clinical isolated multidrug- resistant Gram-negativebacteria Antibiotic KP-2 AB-1 PA-1 ECL-2 ECO-1 Amikacin S R S S SAmpicillin R R R/S / S Aztreonam R R R R R Cefazolin R R R / R CefepimeR R R R R Cefotaxime R R R R R Ceftazidime R R R R R/S Chloramphenicol RR / R S Ciprofloxacin R R R R R Gentamicin R R S R S Imipenem S R S R SLevofloxacin R R R R R Meropenem S R R R S Piperacillin R R R/S R RPolymyxin / S S S / Sulbactam R R / / R/S Sulfanilamide R / R R RTazobactam R R R/S R S Tetracycline R R R R R *R: resistant; S:sensitive.Antibiotics and Chemicals

All experiments were performed in Luria Bertani (LB) medium (EMDmillipore), 2-Phenyl-1, 2-benzisoselenazol-3(2H)-one (ebselen)(Daiichi), 9 antibiotics: ampicillin, carbenicillin, gentamicin,streptomycin, geneticin, kanamycin, chloramphenicol, tetracycline,erythromycin, silver nitrate (Sigma-Aldrich), Methoxypolyethylene glycolmaleimide (MeO-PEG-Mal) (Sigma-Aldrich), Iodoacetamide (IAM)(Sigma-Aldrich), protease inhibitor cocktails (Roche), N-acelytcysteine(NAC) (Sigma-Aldrich), DC™ protein assay (Bio-RAD), E. coli DHB4 TrxR,sheep anti-E. coli Trx1 antibody was from IMCO Corp. (Stockholm, Sweden;http://www.imcocorp.se), Rabbit anti-sheep IgG-HRP (Santa cruz), IgG2amouse monoclonal antibody for glutathione-protein complexes (VIROGEN),4-12% bolt Bis-Tris gel (VWR), all the other reagents were fromSigma-Aldrich.

Antibiotics used in the study with their dosages and primary targetsAntibiotic Abbreviation Dose (μM) used Primary target Ampicillin Amp0/1/2/4 Cell wall formation Carbenicillin Car 0/1/2/4 Cell wallformation Chloramphenicol Chl 0/1/2/4 Protein synthesis, 50S ribosomalsubunit Erythromycin Ery 0/1/2/4 Protein synthesis, 50S ribosomalsubunit Gentamicin Gent 0/1/2/4/80 Protein synthesis, 30S ribosomalsubunit Geneticin Gene 0/1/2/4/80 Protein synthesis, 30S ribosomalsubunit Kanamycin Kan 0/1/2/4/80 Protein synthesis, 30S ribosomalsubunit Streptomycin Str 0/1/2/4 Protein synthesis, 30S ribosomalsubunit Tetracycline Tet 0/1/2/4/80 Protein synthesis, 30S ribosomalsubunitSynergistic Antibacterial Effect of Silver and Antibiotics inCombinations on the Growth of E. coli DHB4

E. coli DHB4 from frozen stock were grown overnight at 37° C., 400 rpm.The overnight culture was diluted 1:100 with 5 ml of LB medium in 15 mltubes and incubated at 37° C. at 400 rpm. Cells were grown until anOD_(600 nm) of 0.4 and were used for antibiotic treatment. Briefly,cells were diluted 1:1,000 into 100 μl of LB medium in 96 micro-wellplates. Serial dilutions of antibiotics 100 μl (0, 1, 2, 4 μM) andsilver nitrate (AgNO₃, 0, 1.25, 2.5, 5, 10, 20, 40, 80 μM) were added tothe individual wells. The minimum inhibition concentration (MIC) wasdetermined as the lowest concentration of drugs that inhibited 90% ofgrowth compared to the untreated cells after 24 h culture at 37° C. Thecultures treated with the same serial dilutions of 100 μl ebselen andsilver nitrate were used as the positive control.

Quantifying Synergy of Ebselen and Silver Using the Bliss Model

Drug synergism was determined using the Bliss Independence Model, whichcalculates a degree of synergy using the following formula:S=(f_(X0)/f₀₀)(f_(0Y)/f₀₀)−(f_(XY)/f₀₀), where f_(XY) refers to thewild-type growth rate in the presence of the combined drugs at aconcentration X, for one of the drugs, and Y for the other; f_(X0) andf_(0Y) refer to the wild-type growth rates in the presence of theindividual drugs at a concentration of X and Y, respectively; f₀₀ refersto the wild-type growth rate in the absence of drugs; and S correspondsto the degree of synergy, a parameter that determines a synergisticinteraction for positive values and an antagonistic interaction fornegative ones. Growth rates at different time points are determined bycalculating the slope of the growth or kill curve being analyzed¹⁸.

Measurement of ROS Production

The E. coli DHB4 cells were grown till the absorbance at OD_(600 nm) of0.4 in LB medium, and the bacterial cells were treated with silver andantibiotics in combinations for 10 min. To analyze the amount of ROSproduction in the bacteria, cells were harvested by centrifugation at6,000 rpm for 5 min and thoroughly washed 3 times with PBS, and stainedwith 5 μM H₂DCF-DA for 20 min. After the incubation, cells were spundown and re-suspended in PBS, and the ROS production was quantified byflow cytometry (CyAnadp, Beckman coulter).

Measurement of H₂O₂ Production

The E. coli DHB4 cells were grown till the absorbance at OD_(600 nm) of0.4 in LB medium, and the bacterial cells were treated with silver andantibiotics in combinations for 10 min. Cells were harvested bycentrifugation at 6,000 rpm for 5 min and thoroughly washed 3 times withPBS, and sonicated for 10 s. In the presence of 50 μM Amplex® Redreagent, 0.1 U/mL HRP in 50 mM sodium phosphate buffer, pH 7.4, 50 μlsamples were incubated for 30 minutes at room temperature protected fromlight and detected with absorbance at 560 nm (Molecular Probes, Eugene,Oreg.).

Measurement of Trx/TrxR Activities and GSH Amount in Antibiotics andSilver Treated E. coli Cell Lysates

E. coli DHB4 cells were grown till the absorbance at OD_(600 nm) of 0.4in LB medium, and the bacterial cells were treated with 80 μMantibiotics and 5 μM AgNO₃ for 10 and 60 min, respectively. The culturestreated with 80 μM ebselen and 5 μM AgNO₃ were used as the positivecontrol. Cells were harvested by centrifugation at 6,000 rpm for 5 minand thoroughly washed 3 times with PBS, then cells were re-suspended inlysis buffer (25 mM Tris·HCl, pH 7.5, 100 mM NaCl, 2.5 mM EDTA, 2.5 mMEGTA, 20 mM NaF, 1 mM Na₃VO₄, 20 mM sodium ß-glycerophosphate, 10 mMsodium pyrophosphate, 0.5% Triton X-100) containing protease inhibitorcocktail and lysed by sonication. The cell lysates were obtained bycentrifugation at 13,000 rpm for 20 min and the protein concentrationswere measured by the Lowry protein assay.

E. coli DHB4 TrxR activity in cell extracts was measured by a DTNBreduction activity assay²³. The experiments were performed with 96micro-well plates in the solution containing 50 mM Tris·HCl (pH 7.5),200 μM NADPH, 1 mM EDTA, 1 mM DTNB, in the presence of 5 μM E. coliTrx1. The absorbance at 412 nm was measured for 5 min with a VERSAmicro-well plate reader and the slope of initial 2 min was used torepresent TrxR activity. The Trx activity was detected by this methodcoupled with 100 nM E. coli TrxR instead of 5 μM E. coli Trx in thereaction mixture. To measure GSH levels, 25 μg of the cell lysates wasadded in the solution containing 50 nM GR, 50 mM Tris·HCl (pH 7.5), 200μM NADPH, 1 mM EDTA, 1 mM DTNB. The absorbance at 412 nm was measuredfor 5 min.

Trx1 Redox State in E. coli Treated by Silver and Antibiotics inCombinations

E. coli DHB4 cells were grown till the absorbance at OD_(600 nm) of 0.4in LB medium, and the bacterial cells were treated with 80 μMantibiotics and 5 μM AgNO₃ for 10 and 60 min, respectively. The culturestreated with 80 μM ebselen and 5 μM AgNO₃ were used as the positivecontrol. Western blotting was performed to detect the Trx1 redox stateof the treated E. coli cells. The cells were harvested by centrifugationat 6,000 rpm for 5 min and thoroughly washed 3 times with PBS, andprecipitated the protein with 5% TCA in 1.0 ml. The precipitates werewashed with 1 ml pre-ice-cold acetone for 3 times and dissolved in 50 mMTris·HCl (pH 8.5) with 0.5% SDS containing 15 mM MeO-PEG-Mal at 37° C.for 2 h. Proteins were obtained by centrifugation at 13,000 rpm for 20min to remove the pellets, and the protein concentration was measured bythe Lowry protein assay. Proteins were incubated with SDS-loading bufferat 90° C. for 10 min, and then separated on the 4-12% bolt Bis-Tris gelwith MES running buffer (150 V, 40 min). The redox state of Trx1 wasdetected with sheep anti-E. coli Trx1 antibody at 1:1000 dilution,followed by the detection of Chemiluminescence Reagent Plus.

Proteins S-Glutathionylation in E. coli Treated by Silver andAntibiotics in Combinations

Total protein S-glutathionylation of antibiotics and AgNO₃ incombinations treated E. coli cells were also detected by Westernblotting. E. coli DHB4 cells were grown till the absorbance atOD_(600 nm) of 0.4 in LB medium, and the bacterial cells were treatedwith 80 μM antibiotics and 5 μM AgNO₃ for 10 and 60 min, respectively.The cultures treated with 80 μM ebselen and 5 μM AgNO₃ were used as thepositive control. Cells were washed 3 times, and re-suspended in lysisbuffer (25 mM Tris·HCl, pH 7.5, 100 mM NaCl, 2.5 mM EDTA, 2.5 mM EGTA,20 mM NaF, 1 mM Na₃VO₄, 20 mM sodium ß-glycerophosphate, 10 mM sodiumpyrophosphate, 0.5% Triton X-100, protease inhibitor cocktail)containing 50 mM IAM. After lysed by sonication, the cell lysates wereobtained by centrifugation at 13,000 rpm for 20 min. Proteinconcentration was measured by Lowry protein assay, and Western blottingassay was performed as described above with IgG2a mouse monoclonalantibody (VIROGEN, 101-A/D8) for S-glutathione-protein complexes.

Antibacterial Effects of Antibiotics and Silver on the Growth ofClinical Isolated MDR Gram-Negative Bacteria

Five clinical isolated MDR Gram-negative strains were grown until anOD_(600 nm) of 0.4, and were diluted 1:1,000 into 100 μl LB medium in 96micro-well plates. Serial dilutions of 100 μl antibiotics (0, 1, 2, 4μM) and AgNO₃ (0, 1.25, 2.5, 5, 10, 20, 40, 80, 160 μM) in combinationswere added to the individual wells. The MIC was determined after 16 hculture at 37° C. The cultures treated with the same serial dilutions of100 μl ebselen and silver nitrate were used as the positive control.

Statistical Analysis

Mean, Standard Deviation (SD) and t-test (two tails, unpaired)significances were calculated in Grap Pad Prism Software. *: p<0.05, **:p<0.01, ***: p<0.001.

TABLE 1 MIC of Silver (μM) in the presence of ebselen against differentmultidrug-resistant Gram-negative species MIC of silver (μM) in thepresence of ebselen against Ebselen multidrug-resistant Gram-negativespecies Others (μM) KP-1 KP-2 AB-1 AB-2 PA-1 PA-2 ECL-1 ECL-2 ECO-1ECO-2 ECO-3 ECO-4 0 80 80 80 80 80 80 80 80 40 80 40 40 1 80 40 80 80 8080 40 40 20 80 40 20 2 40 20 40 40 20 40 20 40 10 40 20 10 4 10 20 10 2020 20 20 10  5 10 10  5 KP-1: Klebsiella pneumoniae (K. pneumoniae)subsp. pneumoniae 13#; KP-2: K. pneumoniae subsp. pneumoniae 0322#;AB-1: Acinetobacter baumannii (A. baumannii) H#; AB-2: A. baumannii0361#; PA-1: Pseudomonas aeruginosa (P. aeruginosa) 1298#; PA-2: P.aeruginosa 0009#; ECL-1: Enterobacter cloacae (E. cloacae) 0431#; ECL-2:E. cloacae 2301#; ECO-1: Escherichia coli (E. coli) 1139#; ECO-2: E.coli 2219#; ECO-3: E. coli ZY-1; ECO-4: MG1655 (ATCC 700926).

TABLE 2 Clinical isolated multidrug-resistant Gram-negative strainsStrain Description KP-1 Klebsiella pneumonia (K. pneumonia) subsp.pneumonia 13^(#) KP-2 K. pneumonia subsp. pneumonia 0322^(#) AB-1Acinetobacter baumannii (A. baumannii) H^(#) AB-2 A. baumannii 0361^(#)PA-1 Pseudomonas aeruginose (P. aeruginose) 1298# PA-2 P. aeruginose0009# ECL-1 Enterobacter cloacae (E. cloacae) 0431# ECL-2 E. cloacae2301# ECO-1 Escherichia coli (E. coli) 1139^(#) ECO-2 E. coli 2219^(#)WZ11 E. coli ZY-1 MG1655 ATCC 700926

TABLE 3 Drug sensitivity of clinical isolated multidrug-resistantGram-negative strains Antibiotic KP-1 KP-2 AB-1 AB-2 PA-1 PA-2 ECL-1ECL-2 ECO-1 ECO-2 ECO-3 Amikacin R S R R S R S S S S S Ampicillin R R RR R/S R / / S R S Aztreonam R R R R R R R R R R S Cefazolin R R R R R R/ / R R S Cefepime R R R R R R R R R R S Cefotaxime R R R R R R R R R RS Ceftazidime R R R R R R R R R/S R S Chloramphenicol R R R R / R R R SS S Ciprofloxacin R R R R R R R R R R R Gentamicin R R R R S R R R S R SImipenem R S R R S / S R S S S Levofloxacin R R R R R R R R R R /Meropenem R S R R R R/S S R S S S Piperacillin R R R R R/S R R R R R SPolymyxin / / S S S S R S / / / Sulbactam R R R / / / / / R/S R SSulfanilamide S R / S R R R R R R R Tazobactam R R R R R/S R S R S R/S STetracycline S R R R R R R R R S S *R: resistant; S: sensitive.

TABLE 4 MIC of silver (μM) in the presence of ebselen against E. coliDHB4 mutants MIC of silver (μM) in the presence of ebselen againstEbselen Escherichia coli DHB4 redox mutants (μM) WT trxA⁻ trxB⁻ trxC⁻trxA⁻B⁻C⁻ oxyR⁻ gshA⁻ trxA⁻gshA⁻ gor⁻ gor⁻grxA⁻B⁻C⁻ grxA⁻trxA⁻ 0 40 4040 40 40 20 20 20 40 20 20 1 10 10 10 10 10  5  5  5 10 10 10 2  5  2.5 2.5  5  2.5  1.25  2.5  2.5  5  2.5  2.5 4  1.25  1.25  0.625  1.25 1.25  0.625  0.625  0.625  1.25  0.625  0.625

TABLE 5 MIC of ebselen (μM) in the presence of silver against E. coliDHB4 mutants MIC of ebselen (μM) in the presence of ebselen againstEscherichia coli DHB4 redox mutants Silver trxA⁻ trxA⁻ gor⁻grxA⁻ grxA⁻(μM) WT trxA⁻ trxB⁻ trxC⁻ B⁻C⁻ oxyR⁻ gshA⁻ gshA⁻ gor⁻ B⁻C⁻ trxA⁻  0 80 80  80  80  80  40  40  40  80  40  40   0.625 8 8 8 8 8 4 4 4 8 8 8 1.25 4 4 2 4 4 2 2 2 4 2 2  2.5 4 2 2 4 2 2 2 2 2 2 2  5 2 1 1 2 1 1 11 1 1 1 10 1 1   0.5 1   0.5   0.5   0.5   0.5 1   0.5   0.5 20   0.5  0.5   0.5   0.5   0.5 0 0 0   0.5 0 0 40 0 0 0 0 0 0 0 0 0 0 0

TABLE 6 Escherichia coli DHB4 redox phenotypes Strain Genotype Wild typeDHB4 (F′ lac-pro lacI^(Q)/Δ(ara-leu)7697 araD139 ΔlacX74 galE galK rpsLphoR Δ(phoA)PvuII ΔmalF3 thi) trxA⁻ DHB4 ΔtrxA trxB⁻ DHB4 trxB::KantrxC⁻ DHB4 ΔtrxC trxA⁻trxB⁻trxC⁻ DHB4 ΔtrxA ΔtrxC trxB::Kan nadB::TnoxyR⁻ DHB4 oxyR::Kan gshA⁻ DHB4 gshA20::Kan trxA⁻gshA⁻ DHB4 ΔtrxAgshA20::Kan gor⁻ DHB4 gor522 . . . mini-Tn10Tc gor⁻grxA⁻B⁻C⁻ DHB4gor522gxA::Kan grxB::Kan mini-Tn10Tc grxC::Cm grxA⁻trxA⁻ DHB4 ΔtrxAgrxA::Kan

TABLE 7 Drug sensitivity of clinical isolated E. coli ZY-1 AntibioticZY-1 Amikacin S Ampicillin S Aztreonam S Cefazolin S Cefepime SCefotaxime S Ceftazidime S Chloramphenicol S Ciprofloxacin S GentamicinS Imipenem S Levofloxacin S Meropenem S Piperacillin S Polymyxin /Sulbactam S Sulfanilamide R Tazobactam S Tetracycline R

TABLE 8 Analysis of blood samples from mice treatment with or withoutsilver and ebselen Vehicle Ebselen + Ag PBS 6h 24h 48h 6h 24h 48h 6h 24h48h ALT (U/L) 32.7 ± 9.3 24.0 ± 3.6 25.33 ± 4.0  79.0 ± 41.0 31.7 ± 6.425.8 ± 5.62 28.7 ± 6.4 25.3 ± 7.2 28.3 ± 17.0 AST (U/L) 130.7 ± 46.6108.0 ± 5.0  106.67 ± 38.1  300.7 ± 15   145.3 ± 28.6 102.5 ± 53.4 113.3 ± 6.0  110.3 ± 23.5 88.7 ± 7.0  BUN (mmol/L)  5.3 ± 1.14  5.9 ±0.93 6.74 ± 2.0   6.0 ± 0.71 6.53 ± 0.8 5.74 ± 0.5   4.6 ± 0.8  7.2 ±0.6 5.8 ± 1.5 CRE (μmol/L)  29.3 ± 2.52  37.7 ± 1.53 29.7 ± 2.08 23.0 ±1.7  39.0 ± 2.6 30.0 ± 5.5  23.0 ± 3.5 26.7 ± 2.3 28.3 ± 5.9  TBIL(μmol/L)  0.10 ± 0.17 0 0 0.43 ± 0.40 0 0 0.40 ± 0.5 0 0 WBC (10⁹/L)3.29 2.52 3.50 4.36 3.27 2.21 3.12 3.13 2.38 Neu# (10⁹/L) 1.46 0.69 0.842.69 1.20 0.52 0.82 0.90 0.55 Lym# (10⁹/L) 1.72 1.72 2.57 0.07 2.39 1.572.2 1.98 1.78 Mon# (10⁹/L) 0.07 0.05 0.03 1.57 0.05 0.01 0.02 0.06 0.04Eos# (10⁹/L) 0.02 0.06 0.06 0.03 0.07 0.01 0.08 0.18 0.01 Bas# (10⁹/L)0.02 0 0 0 0.01 0 0 0.01 0 IMG# (10⁹/L) 0 0 0.01 0.02 0.01 0 0 0.04 0Neu % (%) 44.3 27.3 24.1 61.7 32.2 23.5 26.3 28.7 23.3 Lym % (%) 52.368.4 73.3 1.6 64.5 75.3 70.4 63.4 74.5 Mon % (%) 2 2 0.9 35.9 1.3 0.50.6 1.9 1.7 Eos % (%) 0.7 2.2 1.7 0.7 1.8 0.5 2.6 5.7 0.4 Bas % (%) 0.70.1 0 0.1 0.2 0.2 0.1 0.3 0.1 IMG % (%) 0 0.1 0.2 0.3 0.3 0.1 0 1.4 0PLT (10⁹/L) 574 912 956 566 602 744 605 187 928 MPV (fL) 7.7 6.7 6.5 7.37.1 6.1 7.0 7.9 6.1 PDW 14.8 14.8 14.7 14.9 15 14.7 15 15.4 14.6 PCT (%)0.442 0.613 0.62 0.413 0.428 0.456 0.422 0.147 0.566 P-LCC (10⁹/L) 76 7265 66 61 39 62 37 48 P-LCR (%) 13.2 7.9 6.8 11.7 10.1 5.3 10.3 19.7 5.1RBC (10¹²/L) 9.26 8.56 8.35 7.95 7.25 8.21 8.31 8.91 7.79 HGB (g/L) 145129 127 124 112 130 133 141 122 HCT (%) 48.7 43.3 42 41.8 38 44.8 44.646.6 41.2 MCV (fL) 52.7 50.6 50.3 52.5 52.4 54.5 53.7 52.3 52.8 MCH (pg)15.77 15.1 15.2 15.6 15.5 15.9 16.0 15.8 15.6 MCHC (g/L) 297 299 301 298296 291 297 302 296 RDW-CV (%) 18.1 18.2 18.1 20.2 16.3 21.6 17.8 21.218.1 RDW-SD (fL) 33.2 31.9 32.0 36.7 29.5 41.1 33.3 38.7 33.5 *ALT:alanine transaminase; AST: aspartate aminotransferas; BUN: blood ureanitrogen; CRE: Creatinine; TBIL: total bilirubin; WBC: white blood cellcount; Neu: neutrophil; Lym: lymphocyte; Mon: monocyte; Eos: eosinophil;Bas: basophil; IMG: immunoglobulin; PLT: platelets; MPV: mean plateletvolume; PDW: platelet distribution width; PCT: plateletocrit; P-LCC:large platelet count; P-LCR: large platelet ratio; RBC: red bllod cellcount; HGB: hemoglobin; HCT: hematocrit; MCV: mean corpuscular volume;MCH: mean corpuscular hemoglobin; MCHC: mean corpuscular hemoglobinconcentration; RDW-CV: red cell distribution width coefficient ofvariation; RDW-SD: red cell distribution width standard deviation. Dataare means ± s. d. of three independent experiments.

TABLE 9 Antibacterial effect as MIC of nine antibiotics and silver onwild type E. coli DHB4 Silver Beta-lactams Aminoglycosides TetracyclineMacrolides Synthesis Control (μM) Amp Car Genta Strep Gene Kana TetraEry Chlor Ebse 0 40 40 40 / 40 40 40 40 40 40 1 40 40 20 / 40 20 20 4040 10 2 40 40   2.5 / 10   2.5 10 40 40  5 4 40 40    1.25 40   2.5   1.25    1.25 40 40    1.25 Ampiciline: Amp; Carbenicillin: Car;Gentamycin: Genta; Streptomycin: Strep; Geneticin: Gene; Kanamycin:Kana; Tetracycline: Tetra; Erythromycin: Ery;Chloramphenicol: Chlo;Ebse: Ebselen. Streptomycin: 4 + 40, 8 + 40, 16 + 40, no synergisticeffect.

TABLE 10 MIC of silver (μM) in the presence of different antibioticsagainst MDR Gram-negative bacteria Antibiotics Multi-drug ResistantGram-negative Strains (4 μM) KP-2 AB-1 PA-1 ECL-2 ECO-1 Genta 160 80 40160 40 Kana 160 80 80 160 40 Gene 160 80 80 160 40 Tetra 160 80 80 16080 Ebse 40 10 20 10 5

What is claimed is:
 1. A non-biological surface coated with anantibiotic composition, wherein: the antibiotic composition comprises ametal-containing agent, which comprises silver, and an organoseleniumagent that comprises ebselen or ebselen diselenide, and the antibioticcomposition is present in an amount effective for killing or inhibitingone or more bacteria when the one or more bacteria contact thenon-biological surface.
 2. The non-biological surface of claim 1,wherein the metal-containing agent further comprises copper, zinc,mercury, tin, lead, bismuth, cadmium, cerium, chromium, thallium, or anycombination thereof.
 3. The non-biological surface of claim 1, whereinthe metal-containing agent comprises silver nitrate.
 4. Thenon-biological surface of claim 1, wherein the silver is silver nitrate,silver acetate, silver benzoate, silver carbonate, silver iodate, silveriodide, silver lactate, silver laurate, silver oxide, silver palmitate,silver protein, silver sulfadiazine, or any combination thereof.
 5. Amethod of making the non-biological surface of claim 1, the methodcomprising coating the non-biological surface with the antibioticcomposition comprising the metal-containing agent comprising silver andthe organoselenium agent comprising ebselen or ebselen diselenide. 6.The non-biological surface of claim 1, wherein the non-biologicalsurface is a surgical equipment surface.
 7. The non-biological surfaceof claim 1, wherein the non-biological surface is on or in a jointprosthesis, a dental implant, a catheter, or a cardiac implant.
 8. Thenon-biological surface of claim 1, wherein the organoselenium agent isebselen.
 9. The non-biological surface of claim 1, wherein theorganoselenium agent is ebselen diselenide.
 10. The non-biologicalsurface of claim 1, wherein the metal-containing agent comprising silverand the organoselenium agent are present in a molar ratio ranging fromabout 1:2 to about 1:20.
 11. The non-biological surface of claim 10,wherein the metal-containing agent comprising silver and theorganoselenium agent are present in a molar ratio ranging from about:1:4, 1:8, or 1:16.
 12. The non-biological surface of claim 10, whereinthe organoselenium agent is ebselen.
 13. The non-biological surface ofclaim 10, wherein the organoselenium agent is ebselen diselenide. 14.The non-biological surface of claim 1, wherein the non-biologicalsurface coated with the antibiotic composition exhibits an IC₅₀ value ofabout 50 nM or lower to one or more Gram-negative bacteria.
 15. Thenon-biological surface of claim 14, wherein the one or moreGram-negative bacteria comprises K. pneumonia, A. baumannii, P.aeruginosa, E. cloacae, E. coli, or any combination thereof.
 16. Thenon-biological surface of claim 14, wherein the organoselenium agent isebselen.
 17. The non-biological surface of claim 14, wherein theorganoselenium agent is ebselen diselenide.
 18. The method of claim 5,wherein the organoselenium agent is ebselen.
 19. The method of claim 5,wherein the organoselenium agent is ebselen diselenide.